JP2013240352A - Long-time monitoring method using photoprotein and analysis method - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method of monitoring for a long time a living sample using luminescence from a photoprotein.SOLUTION: A sample of the thickness of 300 μm or more, into which a photoprotein appropriate for the item to be monitored has been transferred under such treating conditions as not injuring the biological activity thereof, is chemically induced with the use of a substrate component, the luminescence induction and image picking thereof are continuously performed during a desired monitoring period to acquire a plural number of optical images corresponding to different times with an image picking volume of less than 300 μm from the surface of the sample and a single cell level, and the plural number of optical images are displayed on a monitor.

Description

  The present invention relates to a monitoring method and an analysis method for detecting a biological activity in a sample such as a cell or tissue for a long period or continuously without damaging the activity as much as possible. The present invention also includes an apparatus for executing the method and software for causing the method by the apparatus to function.

  In research in the fields of biology and medicine, techniques for detecting the biological activity of biological samples such as cells by reporter assays have been widely used. Reporter assays can be used to visualize biological activities that cannot be visually examined. In conventional clinical tests, only biological substances (nucleic acid, blood, hormones, proteins, etc.) to be examined from biological samples are isolated by various separation methods, and the amount and activity of the isolated biological substances are determined as reagents. And reacted. However, in living organisms, the interaction between various biological materials shows true biological activity. In recent years, when researching or developing medical drugs, drugs that most effectively act on biological activity in living biological samples have become critical conditions. In reporter assays for living biological samples, there is a growing need to image biological samples and biological substances to be examined and observe dynamic changes inside and outside the biological samples over time.

  Specifically, in research fields that utilize observation using luminescence (bioluminescence, chemiluminescence) or fluorescence as a reporter substance, time lapse or time lapse is used to capture the dynamic expression of protein molecules in a sample. There is a need for video imaging. At present, dynamic changes are observed using an image picked up from a fluorescent sample (for example, observation of a moving image of one protein molecule using fluorescence). In the case of imaging a fluorescent sample, the amount of light emitted from the fluorescent sample decreases with the lapse of time by continuing to irradiate excitation light, so that a stable image that can be used for quantitative evaluation can be taken over time. However, it was possible to take a clear, high spatial resolution image with a short exposure time. On the other hand, in the time-dependent observation of dynamic changes by images for a luminescent sample, the luminescence from the luminescent sample is extremely small, so a CCD camera equipped with an image intensifier was used to observe the luminescent sample. It was done. In the case of imaging of a luminescent sample, it was not necessary to irradiate excitation light, so that stable images that could be used for quantitative evaluation could be taken over time.

  Until now, in the observation of a luminescent sample, the amount of luminescence from the luminescent sample has been measured. For example, in the observation of a cell into which a luciferase gene has been introduced, the amount of luminescence from the cell due to the luciferase activity was measured in order to investigate the intensity of the luciferase gene expression (specifically, the expression level). . The amount of luminescence from the cells due to luciferase activity is measured by first reacting a cell lysate in which cells are lysed with a substrate solution containing luciferin, ATP, magnesium, etc., and then reacting with the substrate solution. The amount of luminescence from was quantified with a luminometer using a photomultiplier tube. That is, the amount of luminescence was measured after cell lysis. Thereby, the expression level of the luciferase gene at a certain time point could be measured as an average value of the whole cell. Here, methods for introducing a luminescent gene such as a luciferase gene into a cell as a reporter gene include, for example, a calcium phosphate method, a lipofectin method, an electroporation method, and the like, and each method is properly used depending on the purpose and the type of cell. Yes. In addition, when investigating the intensity of luciferase gene expression in cells transfected with a luciferase gene as a reporter gene using the amount of luminescence from the cell due to luciferase activity as an indicator, the target luciferase gene is introduced upstream or downstream of the luciferase gene introduced into the cell. By linking DNA fragments, it is possible to examine the effect of the DNA fragment on the transcription of the luciferase gene. In addition, genes such as transcription factors that are thought to affect the transcription of the luciferase gene to be introduced into cells are linked to the expression vector. By co-expressing with the luciferase gene, the effect of the gene product of the gene on the expression of the luciferase gene can be examined.

  In addition, in order to capture the expression level of the luminescent gene over time, it is necessary to measure the amount of luminescence from living cells over time. In order to measure the amount of luminescence from living cells over time, first add the function of a luminometer to the incubator that cultivates the cells, and then quantitate the amount of luminescence from the whole cell population cultured at regular intervals with the luminometer. The procedure was to do. As a result, it was possible to measure an expression rhythm with a certain periodicity, and thus, it was possible to capture changes over time in the expression level of the luminescent gene in the entire cell. In order to sufficiently secure the measurement sensitivity by the luminometer, a general-purpose substrate solution such as firefly luciferin was treated at a concentration of 1 mM or more. In this concentration setting, since the cells are taken out and processed, they are not processed in a living state that can be returned to the culture environment.

  On the other hand, when the expression of the luminescent gene is transient, the expression level in individual cells varies greatly. For example, even in the case of cloned cultured cells such as HeLa cells, the response of drugs via receptors on the cell membrane surface may vary among individual cells. That is, several cells may be responding even if the response as a whole cell is not detected. For this reason, when the expression of the luminescent gene is transient, it is important to measure the amount of luminescence from individual cells over time rather than from the whole cell. The time-dependent measurement of the amount of light emitted from living individual cells using a microscope shows that the light emitted from each cell is extremely weak, so it can be exposed for a long time with a cooled CCD camera at a liquid nitrogen temperature level, or an image intensifier. Or a photon counting device. As a result, it was possible to capture changes over time in the expression level of the luminescent gene in individual living cells. Regardless of the culture period, the reagent conditions for luminescence are not changed.

  In the above description, for example, a method and an apparatus for analyzing gene expression using a fluorescent protein as a reporter gene are disclosed in Patent Document 1 (Japanese Translation of PCT International Publication No. 2004-5000576). A method and apparatus for analyzing gene expression by bioluminescence using a luminometer is disclosed in Patent Document 2 (Japanese Patent Laid-Open No. 2005-1118050).

Japanese translation of PCT publication No. 2004-500576 JP 2005-1118050 A

  However, when imaging a weakly luminescent sample, the amount of light emitted from the luminescent sample is extremely small, so it cannot be viewed with the naked eye, and the amount of light must be accumulated using a storage-type imaging means such as a CCD. Therefore, there is a restriction that an image cannot be generated. Moreover, in the cell group constituting a single cell or tissue, the weak light generated from each cell is too weak, so that the exposure time required for taking a clear image becomes long. there were. That is, since the imaging time interval is limited by the amount of light per unit time, when imaging a luminescent sample with weak light emission, a clear image can be taken over time at a long time interval, for example, at an interval of 60 minutes. Even if it was possible, there was a problem that imaging could not be performed in real time with a short exposure time of about 10 to 30 minutes, and thus with an exposure of 1 to 5 minutes. In particular, when living cells are exposed for a long time (for example, 50 minutes or longer), the cells themselves may move even on a support such as a culture vessel and a clear image may not be formed. Generally, in order to perform an analysis using an image, it is necessary to recognize an accurate contour. Therefore, the analysis result may be inaccurate when the image is unclear.

  The present invention has been made in view of the above problems, and even with a sample into which a photoprotein that generates weak light that cannot be seen with the naked eye is introduced, there is almost no damage to the sample, and desired monitoring and analysis are possible. It aims to provide a simple method. It is another object of the present invention to provide a method that enables single-cell level monitoring and analysis of a sample containing cells. It is another object of the present invention to provide a method that combines a luminescent sample imaging method capable of capturing a sample that emits light with various amounts of light by a photoprotein with a clear exposure time and in real time while being a clear image.

  The inventors have found that when a live sample containing cells is treated with a high concentration substrate solution to sharpen the image, inaccurate results can occur during the long-term monitoring or analysis process. Such a result was also found when the sample was prepared with a small thickness. It has been found that the processing conditions for performing clear and sensitive monitoring and / or analysis contradict the processing conditions for performing long-term and accurate monitoring and / or analysis. As a result of intensive studies, the inventors have realized that the above conflicting problems can be solved unexpectedly by associating the sample processing conditions with the imaging conditions.

That is, in order to solve the above-described problems, the method of the first example of the present invention is a method for monitoring a living sample for a long time by luminescence based on a photoprotein, under a processing condition that does not impair biological activity. The sample into which the photoprotein according to the item to be introduced is chemically induced by the substrate component, and during the desired monitoring period, the induction and imaging of the luminescence are continuously performed, and set according to the processing conditions. A long-term monitoring method using a photoprotein characterized in that an optical image corresponding to a plurality of different times is obtained under a given imaging condition, and the plurality of optical images are displayed on a monitor according to an output condition corresponding to the imaging condition. is there. The second example is a monitoring method characterized in that, in the method of the first example , when the monitoring period is several days or more, the concentration of the substrate is 700 μM or less. The third example is a monitoring method characterized in that, in the method of the first example , when the monitoring period is less than several days, the concentration of the substrate is 800 μm or more and 1 mM or less. The fourth example is a monitoring method characterized in that, in the method of the first example , the substrate concentration is 200 μM or more.

In addition, the fifth example was invented by paying attention to the volume of the sample as a processing condition. In the method of the first example , the fifth example was applied when monitoring a single cell level with a sample containing cells into which a photoprotein was introduced, An optical image based on light emission having a sufficient thickness that does not impair reactivity with a substrate component for generating weak light emission in cells and a drug component that may affect light emission of the photoprotein. This is a monitoring method characterized by acquiring an optical image corresponding to an imaging volume that is thin enough to identify individual cells. The sixth example is a monitoring method according to the fifth example , wherein the sample is composed of a group of cells arranged in a two-dimensional substantially constant state. The seventh example is a monitoring method according to the sixth example , wherein the cell group has a density such that the cells do not overlap on the optical path for imaging. The eighth example is a monitoring method according to the sixth example or the seventh example , wherein the cell group is prepared as a sliced section or a sheet-like cell. The ninth example is a monitoring method characterized in that, in the method of any one of the fifth to eighth examples , the sample has a thickness of 150 μm or less, preferably 100 μm or less, particularly less than 85 μm. The tenth example is a monitoring method characterized in that in the ninth example , the thickness is 40 μm or more, preferably 50 μm or more.

In addition, according to the method according to the eleventh example , in the method according to the fifth example , the test sample is a thick biological tissue (for example, a brain such as the upper crossing nucleus, an embryo, a small fish such as a zebrafish, a mouse, etc. Biological specimens of biological tissues such as frogs, frogs (especially Xenopus laevis), and other organs (eg, hands, feet, hair roots, leaves, flower stems, root hairs) of small animals or plants (eg, Arabidopsis thaliana) In addition, according to the twelfth example , in the method of any of the first to fourth examples , the substrate is firefly luciferin or coelenterazine, so that genetic treatment is performed. It is preferable because it is easy to do.

Further, according to the monitoring method of the thirteenth example, in the method of the first example or the fifth example , the optical imaging is the square of (NA ÷ β) represented by the numerical aperture (NA) and the projection magnification (β). It is preferable that a clear image can be formed at an early stage by using optical conditions including an objective lens having a value of 0.01 or more. Here, the value is more preferably adjusted according to the magnification of the objective lens. In addition, according to the monitoring method of the fourteenth example, the method of the first or thirteenth example includes a step of adjusting the length of the exposure unit time for obtaining one light emission image. According to the monitoring method of the fifteenth example, the method of the fourteenth example further includes a step of dividing the image obtained in the unit time.

The 16th example of how the analysis of the present invention is a method of analyzing long term by luminescence based raw Kita sample luminescence protein, in processing conditions that do not impair the biological activity, luminescence protein corresponding to the item to be analyzed The sample having introduced therein is chemically induced by a substrate component, and during the desired monitoring period, the induction and imaging of the emission are continuously performed, and a plurality of samples are obtained under imaging conditions set according to the processing conditions. It is a long-term analysis method using a photoprotein characterized in that optical images corresponding to different times are obtained, and information necessary for analysis is obtained by performing image processing on the optical image according to the imaging conditions. Here, in the seventeenth example , in the method of the sixteenth example , the concentration of the substrate component as the processing condition is 600 μM or less, and the imaging condition is represented by a numerical aperture (NA) and a projection magnification (β). The analysis method is characterized in that the exposure time is such that an image based on a photoprotein is obtained using an optical condition including an objective lens whose square value of (NA ÷ β) is 0.01 or more. Furthermore, in Example 18 , in the method of Example 16 or Example 17 , the substrate concentration was present at 200 μM or more, and the imaging condition was set within an exposure time in which the biological activity of the sample did not substantially change. This is an analysis method characterized by

The nineteenth example is applied to a sample further containing a fluorescent component in the sixteenth example , and the information required for the analysis is obtained from each optical image of light emission and fluorescence. Is the method. The fluorescent component is set so as to meet the above-described processing conditions, and may have a concentration that is so small that it is not necessary to apply excessive excitation light. As in the twentieth example , it is preferable that the method of the nineteenth example determines the amount of excitation light according to the time required for acquiring the luminescent image. In addition, the twenty-first example is an analysis method characterized in that the method of the sixteenth or seventeenth example includes a step of adjusting the length of the exposure unit time for obtaining one light-emitting image. The twenty-second example is an analysis method characterized in that the method of the twenty-first example further includes a step of dividing the image obtained in the unit time.

  According to the above monitoring method and analysis method, an apparatus or method having the following characteristics can be included. That is, an apparatus for executing the method of the present invention prepares a cell or a biological sample containing the cell as a specimen in a state in which an image can be obtained, and weak optics resulting from biological activities from a large number of the specimens. In cooperation with weak light image acquisition means that accumulates the target data and acquires image information that can be analyzed, it recognizes individual cells by morphologically analyzing the weak light image and corresponds to the recognized individual cells The image analysis is performed to comprehensively evaluate the light intensity of the weak light. Here, in the case where the weak light image acquisition means has a configuration for continuously or intermittently acquiring weak light images at different desired elapsed times, the light intensity per time for one or more identical cells. By comprehensively analyzing the above, it becomes possible to comprehensively evaluate, for example, the temporal gene activity pattern and the response pattern of intracellular substances due to drugs and the like. In addition, the cells that do not exhibit a predetermined light intensity or light distribution among the recognized cells are not subjected to exhaustive evaluation, so that accurate evaluation can be performed excluding cells that should not be analyzed. Further, by calculating the total value or the average value of the light intensities of all the cells subjected to the image analysis, it becomes possible to perform the evaluation of the analyzed whole cells in addition to the evaluation of the individual cells. Further, by classifying the cells into the same or different cell groups according to the light intensity and / or light intensity pattern of two or more cells subjected to the image analysis, the activity can be evaluated for each analyzed pattern. In some cases, it becomes possible to examine details of activity or activity change for each cell having a different pattern. In particular, in the present invention, as described later, the present invention further includes a step of adjusting the length of the exposure unit time for obtaining one luminescent image, and preferably dividing the image obtained in the unit time. The effects of the invention are made more advantageous.

  In addition, the apparatus of the present invention uses a cell or a biological sample containing the cell as a specimen, a holding means for holding a large number of the specimens in a state in which an image can be acquired, and a weak optical emitted from the biological sample. Weak light image acquisition means for accumulating data and acquiring image information that can be analyzed, and recognizing individual cells by morphologically analyzing the weak light image and detecting weak light corresponding to the recognized individual cells An image analysis means for comprehensively evaluating the light intensity is provided. Here, since the holding means has a configuration in which a plate in which a plurality of wells are integrated is held so as to be addressable, evaluation between the plurality of wells is performed in the same field of view or in a predetermined order. It becomes possible to compare and correlate the results of activity evaluation with different samples or different drugs. In this case, the holding means may be configured to hold a plurality of independent containers in an addressable manner, and is not limited to the field of view of the image acquisition means, and can evaluate a large number of containers. In addition, by having a control unit that controls to perform evaluation according to the time of image acquisition, analysis of the same cell for each elapsed time, cells of different times (identical or different cells) exhibiting a specific activity Various time analysis such as comparative analysis can be performed. Further, by further including display means for displaying the image analysis result in association with image information, an image corresponding to the result desired to be viewed as an image can be displayed from the analysis result. In addition, since the display means has a configuration in which desired image information is displayed as a moving image, a change in activity relating to one or more desired cells can be observed with a real-time image. As a moving image display, it is more preferable to enhance the sense of reality by superimposing weak light images of the same cell for each time by image processing. Further, a plurality of time-series images related to the same cell may be displayed in parallel (or just partially shifted) by frame advance so that the entire image for each time can be displayed.

  Further, the analysis method in the present invention is a method for analyzing an interaction between cells and tissues, wherein an interaction substance obtained by labeling a luminescent substance as a biological excitation substance on at least one of the interactable substances is provided. Including the steps of optically capturing the light emission resulting from the interaction in a plurality of cells through an imaging field including a plurality of cells, and individually analyzing the plurality of cell images in the same field. It is characterized by. Here, the presence and / or amount of weak light in the individual cell image is preferably correlated with the interaction. It is also preferred that the biological excitation material includes a component that provides bioluminescence. More preferably, the component that provides bioluminescence uniformly contains a substrate for the luminescent reaction.

  Moreover, it is preferable that the analysis method of the present invention includes a step of evaluating an interaction with a chemical stimulating substance that affects the activity of the biologically excited substance. Preferably, the method further includes a step of evaluating an interaction with a physical stimulus that affects the activity of the biologically excited substance. Further, it is more preferable that the biological sample contains living cells. Moreover, it is preferable that the said analysis process includes the measurement process which measures several cells in the same visual field statistically. Here, it is more preferable that the measurement step includes a step of calculating at least the number of cells that generate detectable luminescence. Further, it is more preferable that the counting step includes a step of sorting by group according to the amount of luminescence for each cell. In any case, it is preferable to include a step of expressing the result information by the measurement step in association with the cell image so as to be visible. Instead of the expressing step, the imaging step is executed at a number of different timings, and the result information from the measuring step is output in real time along the time axis or synchronized along the activity curve of the luminescence reaction. This is also preferable when it includes a step of outputting the output.

  Further, the analysis method of the present invention is a method for analyzing an interaction between cells and tissues, wherein an interaction substance obtained by labeling a luminescent substance as a biological excitation substance on at least one of the interactable substances is provided. The same cell or multiple different parts of the same tissue can be present at the same time, and the resulting luminescence can be optically imaged by the imaging field including the cell or tissue, The method includes a step of analyzing based on image information obtained by imaging information related to the part.

  Further, the analysis method of the present invention is a method for analyzing an interaction between cells and tissues, wherein an interaction substance obtained by labeling a luminescent substance as a biological excitation substance on at least one of the interactable substances is provided. The same cells or the same tissue exist in a specific place or distribution state of the same cell or the same tissue, and the luminescence as a result of the interaction is optically imaged in time series by the imaging field including the cell or the tissue, The method includes a step of analyzing a change in a place or a distribution state of the luminescent material based on image information obtained by imaging information on a plurality of parts therein.

  Further, the analysis method of the present invention is a method for analyzing interactions inside and outside a cell or tissue, wherein a plurality of types of interacting substances that can interact are labeled with a luminescent substance as a biological excitation substance or the cells or Including the step of optically imaging light emission as a result of interaction in a tissue through an imaging field including the cell or tissue, and analyzing each of the interacting substances based on the captured image information. Features. Here, it is preferable that the plurality of types of interacting substances are separately labeled with luminescent substances having different optical characteristics that are optically distinguishable. In addition, it is preferable to perform chemical or physical stimulation specific to each of the plurality of types of interacting substances at different timings.

  The above method and apparatus may be provided in the form of software for controlling or coordinating a required apparatus configuration or a computer program characterizing the software. In addition, by electrically connecting the method or apparatus of the present invention to a database arranged identically or separately from the apparatus, it is fast and reliable and high quality without being limited by image capacity or analysis information amount. Analysis results can be provided.

  According to the present invention, it is important in practical use to capture a luminescence image of a cell into which a luciferase gene has been introduced with an optical system that satisfies the following conditions. According to the optical experiment by the present applicant, imaging is performed with an optical system in which the square of (NA ÷ β) expressed by the numerical aperture (NA) of the objective lens and the projection magnification (β) is 0.01 or more. Thus, it was proved that an image can be formed only by luminescence generated from a single cell (see Japanese Patent Application No. 2005-267531). Furthermore, the present applicant has proceeded with studies and found that the variation pattern of gene expression is different among a plurality of cells cultured in the same petri dish. Further, according to the optical condition investigated by the inventor, when the optical condition expressed by the square of the numerical aperture (NA) / projection magnification (β) of the objective lens of the imaging apparatus is 0.071 or more. It was found that a cell image that can be imaged within 1 to 5 minutes and can be analyzed is provided. A light emission analysis system that observes these light emission images with a storage-type imaging device under a microscope is called a light emission microscope. The light emission microscope preferably has a light shielding means having an opening / closing lid (or an opening / closing window) for shielding light, and a necessary biological sample is set or exchanged by opening / closing the light shielding means. Depending on the purpose, an operation for chemically or physically stimulating a vessel containing a biological sample may be performed manually or automatically. In the best mode, the luminescence microscope is equipped with a known or unique culture device. The culture apparatus has a function of optimally maintaining the temperature, humidity, pH, outside air component, medium component, and culture solution component so as to enable analysis in the system for a long period of time.

  Therefore, the analysis method of the present invention is a method for analyzing intracellular / extracellular interactions based on image information, wherein at least one of the interactable substances is labeled with a luminescent substance as a biological excitation substance. Including the step of causing the agent to be present in one or more cells, optically imaging the resulting emission of the interaction, and imaging the captured optical information for a single cell, wherein the image representation is an interaction It is characterized by the presence and / or amount of weak light caused by it.

  In the present invention, the following application ranges can be included.

(1) Assay Items In the following wide range of assay items, low invasiveness and direct analysis using weak luminescence can be performed in detail and accurately. For example, gene expression abnormalities (eg determination of gene expression frequency and / or monitoring of fluctuations), internal rhythm disorder related diseases (eg sleep disorders, overwork syndrome, jet lag), temporal pharmacological responses (eg drug sensitivity or drug metabolic activity) Diurnal fluctuations), sunshine response rhythms (eg plant growth rate, phototactic motor activity), chemical response evaluation (drug discovery screening, anticancer drug effect monitoring, post-surgical follow-up of cells (or biological tissues) after transplantation or gene therapy , Water (or serum) stress evaluation), physical stimulus response evaluation (heat shock, electric shock, pressure shock), and developmental biological activity evaluation.

(2) Assay principle Nuclear translocation, receptor receptor signal transduction, nerve cell growth, and biological circadian rhythm. Among these, in the case of using an optical or thermal stimulus in the assay, the conventional fluorescence detection causes an extra light stimulus or a heat increase due to multiple excitation light irradiations, but because it is a cold light in the luminescence, There is almost no influence on the cell by excessive exogenous beam and heat rise.

(3) Usable devices An imaging device (for example, an optical microscope, a photomultiplier type imager (photomal), an image cytometer), and a spectroscopic measurement device (for example, a luminometer, an integrating sphere photometer, a photon counter) can be mentioned. Among these, the image pickup apparatus is preferably a CCD camera (excellent cooling performance when sensitivity reduction due to incubator temperature is a problem in a system integrated with a culture apparatus) rather than image generation by photomultiplier. ) Is preferable in that a clear image quality can be easily obtained in a short time. However, in measuring biological activity, data can be obtained by any of the imaging apparatus and the spectroscopic measurement apparatus. For example, a light emission amount (or light emission intensity) sufficient to show biological activity can be accumulated before an image is generated in the imaging device. Therefore, when a luminescent image is acquired using any one of the imaging means, it is possible to obtain highly sensitive luminescent data using the same luminescent image. When performing continuous or time-lapse (or time-series) analysis, image generation is performed at least once (preferably the first time) of the emission signals among a plurality of different times, otherwise the image is generated. If only light emission data can be repeated preferably for a designated part or partial region without generation, it is possible to further improve the efficiency of evaluation or analysis time. In the present invention, when a spectroscopic measurement device is used, a specific single cell can be obtained by combining with a means (for example, an optical aperture) capable of measurement limited to a specific part or partial region of a biological sample. The received light data that is smaller than the maximum light-receiving area and contains as little extra area as possible can be obtained continuously or over time by a slightly larger small frame or a large frame including a cell population having a certain extent. Photometry may be repeated repeatedly (preferably in time series).

(4) Target of assay (sample)
Examples include cells or tissues derived from eukaryotes and cyanobacteria. Particularly exemplified in medical applications are samples containing cells excised by biopsy from the site to be examined in mammals, particularly humans. In regenerative medicine, at least a part of an artificially modified or synthesized biological sample can be used for the purpose of examining whether biological activity is maintained well. In another aspect, the assay subject of the present invention is not limited to cells or biological tissues derived from animals, but may be cells or biological tissues derived from plants or insects. In the case of bacteria and viruses, the analysis of each part in the container that has not been performed by a conventional luminometer can be targeted. In a luminometer, a large amount of light emission is obtained by stacking innumerable samples (for example, 1 million or more per well) in a container such as a well or a petri dish. In the present invention, individual cells or living tissues can be analyzed by storing them in a container at a density that allows individual cells to be identified. Since individual analysis can include a step of calculating the number of only luminescent cells, an accurate interaction per cell can be evaluated. In particular, according to the present invention, a living cell can be monitored and / or analyzed for a long period of time stably and without damage by treating the sample with a photoprotein that does not require excitation light and can generate weak luminescence over a long period of time. Suitable for

(5) Significant use provided by the present invention One or a plurality of types of luminescence data generated from each individual cell. As an example, a method and apparatus for statistically elucidating that each cell exhibits a different light emission amount or light emission distribution is provided. As another example, a method and apparatus for a multi-assay that elucidates whether different types of luminescence from the same cell occur at the same or different times is provided. As another example, a method for elucidating a plurality of identical or different cell populations classified by the amount of luminescence in the same imaging region, and performing a graphic expression such as a histogram or a normal distribution on the classification results as necessary, and Providing the device. As another example, there is provided a method and apparatus for stably and accurately analyzing various changes in a specific region of a same cell (or tissue) or a region having a certain spread. Provided is a method and apparatus for elucidating a plurality of identical or different cell populations classified by the amount of luminescence in a distribution state imaging region, and performing a graphic expression such as a histogram or a normal distribution on the classification results as necessary.

  In the present invention, it should be noted that, by changing the substrate concentration setting according to the culture period, a luminescent image can be obtained for a long time, and the biological activity of the cells can be maintained for a long time. The inventors have also found that can be achieved simultaneously. Therefore, the present invention includes the following inventions in order to solve the problems that may occur in the culture environment and achieve the object.

  That is, according to the present invention, a living cell is treated with a luminescent component, and the substrate solution for inducing luminescence to the luminescent component is allowed to exist in an appropriate culture environment to cause the cell to emit light, Providing a method for processing live cells for image analysis, characterized in that the substrate concentration is set so as not to impair the biological activity of the cells according to the culture period to be analyzed in the analysis based on the luminescence image of the cells. To do. Moreover, when the culture period is several days or more, the treatment method is characterized in that the concentration of the substrate is 700 μM or less. In addition, when the culture period is less than several days, the treatment method is characterized in that the substrate has a concentration of 800 μm or more and 1 mM or less. The treatment method of the present invention maintains a state in which the biological activity is not lowered by continuing the culture in the substrate concentration range as described above, and always maintains an imageable luminescence amount during the culture period. .

On the other hand, according to the present invention, the living cells are treated with the luminescent component, and the cells are allowed to emit light by causing a substrate solution for inducing luminescence to the luminescent component to exist in an appropriate culture environment, In the analysis based on the luminescence image of the cell, the optical condition where the square value of (NA ÷ β) expressed by the numerical aperture (NA) and the projection magnification (β) is 0.01 or more is from the cell. There is also provided an image analysis method for living cells, characterized by collecting luminescence and setting the substrate concentration to 600 μM or less. Further, since the value of the square of (NA ÷ β) as the optical condition is 0.039 or more, an analysis method using a light emission image in a shorter time is provided. If a luminescent image can be obtained in a short time, it is possible to guarantee a sufficient image quality for accurate image analysis. In addition, according to the same optical conditions, there is an advantage that small and economical imaging can be performed without using an expensive cryogenic cooling type imaging device. When imaging can be performed at a significantly higher speed than before, it is preferable in that image analysis of easily movable cells (neural cells, cyanobacteria, etc.) can be reliably performed. Further, when the value of (NA ÷ β) ² expressed by the numerical aperture (NA) and the projection magnification (β) is 0.071 or more, by using the objective lens in a short time of 1 to 5 minutes, This is preferable because real-time imaging can be performed in any cell analysis.

  Further, the present invention makes it possible to perform image analysis at short time intervals using an objective lens having a high numerical aperture (NA) for luminescence imaging, so that all stimulus responsiveness is not missed. This provides an excellent method in drug discovery and diagnosis. In addition, a luminescent sample [eg, a luminescent protein {eg, a luminescent protein expressed from an introduced gene (eg, luciferase gene)], a luminescent cell or a collection of luminescent cells, Even in the case of tissue samples or luminescent individuals (such as animals and organs), a clear image can be analyzed with a short exposure time and in real time.

  ADVANTAGE OF THE INVENTION According to this invention, the method of performing monitoring and analysis by a photoprotein stably for a long period without impairing the biological activity of a sample can be provided.

FIG. 1 is a diagram showing an outline of an apparatus for carrying out the analysis method of the present invention. FIG. 2 is a photographic drawing of a luminescence image when the luciferin concentration is high and the exposure time is extremely short. FIG. 3 is a photographic drawing of a luminescence image when the luciferin concentration is an intermediate concentration and the exposure time is extremely short. FIG. 4 is a photographic drawing of a luminescence image when the luciferin concentration is low and the exposure time is extremely short. FIG. 5A is a photographic drawing of the luminescence image when the luciferin concentration is low and the exposure time is set longer. FIG. 5B is a graph showing a result of comparison of light emission intensity under each imaging condition. FIG. 6-1 is a photograph of an image showing the result of siRNA inhibition against the pGL3 gene. FIG. 6-2 is a graph showing the results of inhibition of siRNA against the pGL3 gene in two types of dishes. 6-3 is a graph showing the results of analysis over time of RNA interference by the method and reagent kit of Embodiment 1. FIG. FIG. 7-1 is a diagram showing a luciferase-YFP vector (pRSETB) used in the method of embodiment 2. FIG. 7-2 is a graph showing a change with time of emission intensity at each wavelength. FIG. 7-3 is a graph showing changes in the intensity of YFP (546 nm) and Gaussia (478 nm). FIG. 7-4 is a graph showing the ratio of intensity change between YFP (546 nm) and Gaussia (478 nm). FIG. 8-1 is a photographic drawing of an image without a filter before the start of stimulation. FIG. 8-2 is a schematic diagram illustrating an image with and without a filter 3 hours after the start of stimulation. FIG. 9 is a diagram showing changes over time in the luminescence of luciferase and the fluorescence intensity of GFP in the square region in FIG. FIG. 10A is a diagram illustrating a bright-field image when identification is performed in the second embodiment of the third aspect. 10-2 is a diagram illustrating a light emission image when identification is performed in Example 2 of Embodiment 3. FIG. 10C is a diagram illustrating an image obtained by superimposing the images illustrated in FIGS. 10A and 10B. FIG. 11 is a diagram showing the results of analyzing luciferase activity for each elapsed time after stimulation with ATP. 12A is a diagram illustrating a bright-field image when identification is performed in Example 3 of Embodiment 3. FIG. 12-2 is a diagram illustrating a light emission image when identification is performed in Example 3. FIG. 12C is a diagram illustrating an image obtained by superimposing the images illustrated in FIGS. 12A and 12B. FIG. 13 is a diagram showing the results of analyzing luciferase activity for each elapsed time after stimulation with serum. FIG. 14 is a diagram showing the interaction between the Id gene site in the NLuc-Id gene and the MyoD gene site in the MyoD-CLuc gene expressed in cells in the embodiment 4. FIG. 15 is a diagram showing induction using a stimulating substance in cell B in which the NLuc-Id gene (or MyoD-CLuc gene) is expressed. FIG. 16 is a diagram showing GFP staining (left side of the figure) and immunostaining (right side of the figure) of Hela cells expressing a fusion protein containing a transmembrane domain in the embodiment 5. FIG. 17A is a diagram illustrating a bright-field image (left side of the figure) and a luminescence image (right side of the figure) obtained after the reaction with sig pep-Luci-GPI. FIG. 17-2 is a diagram showing a bright-field image (left side of the figure) and a luminescence image (right side of the figure) obtained after the reaction by Luci-GPI. FIG. 18 is a diagram showing the results of measuring a cell lysate obtained by lysing cells with a luminometer.

  Embodiments of a faint light analysis method according to the present invention will be described below in detail with reference to the drawings. Note that the present invention is not limited to the embodiments.

  A configuration of an apparatus for carrying out the method according to the present invention will be described with reference to FIG. FIG. 1 is a diagram showing an example of the configuration of an apparatus for carrying out the luminescent sample imaging method according to the present invention. As shown in FIG. 1, an apparatus for carrying out a luminescent sample imaging method according to the present invention is for imaging a sample 1 to be imaged with a short exposure time, and in real time. The condenser lens 3, the CCD camera 4, and the monitor 5 are included. The apparatus may further include a zoom lens 6 as shown.

Sample 1 is a luminescent sample, for example, a luminescent protein {for example, a luminescent protein expressed from an introduced gene (such as a luciferase gene)}, a luminescent cell, an aggregate of luminescent cells, or a luminescent material. Tissue samples, luminescent organs, and luminescent individuals (animals, etc.). Sample 1 may specifically be a luminescent cell into which a luciferase gene has been introduced. The objective lens 2 has a value of (NA ÷ β) ² expressed by a numerical aperture (NA) and a projection magnification (β) of 0.01 or more. The condenser lens 3 collects light emitted from the sample 1 that has reached through the objective lens 2. The CCD camera 4 is a cooled CCD camera at about 0 ° C. and images the sample 1 through the objective lens 2 and the condenser lens 3. The monitor 5 outputs an image captured by the CCD camera 4.

The value of (NA / β) ² is written on the objective lens 2 and the packaging container (package) of the objective lens 2. The conventional objective lens indicates the lens type (eg “PlanApo”), magnification / NA oil immersion (eg “100 × / 1.40 oil”) and infinity / cover glass thickness (eg “∞ / 0.17”). It had been. However, the objective lens (objective lens 2) of the imaging means according to the method of the present invention includes a lens type (for example, “PlanApo”), magnification / NA oil immersion (for example, “100 × / 1.40 oil”), infinity / In addition to the cover glass thickness (for example, “∞ / 0.17”), an injection opening angle (for example, “(NA / β) ² : 0.05”) is also described.

As described above, in the apparatus for carrying out the luminescent sample imaging method according to the present invention, the objective lens 2 is represented by the numerical aperture (NA) and the projection magnification (β) (NA ÷ β) ² . The value is 0.01 or more. As a result, a luminescent sample (for example, a luminescent protein (for example, a luminescent protein expressed from an introduced gene (for example, luciferase gene)), a luminescent cell, or a collection of luminescent cells, A clear image can be taken with a short exposure time and even in real time even with a sex tissue sample or a luminescent individual (such as an animal or an organ). Specifically, a luminescent cell into which a luciferase gene is introduced can be imaged, and a clear image can be taken with a short exposure time and in real time. In addition, since the objective lens 2 has a larger numerical aperture and a lower magnification as compared with a conventional objective lens, a wide range can be imaged with high resolution by using the objective lens 2. Thereby, for example, a moving luminescent sample, a moving luminescent sample, or a luminescent sample distributed over a wide range can be set as an imaging target. The objective lens 2 is expressed by a numerical aperture (NA) and a projection magnification (β) on the objective lens 2 and / or a packaging container (package) for packaging the objective lens 2 (NA ÷ β). A value of ² (for example, 0.01 or more) was expressed. Accordingly, for example, a person who observes a luminescent image can easily obtain an objective lens suitable for imaging a luminescent sample in a short exposure time and in real time by confirming the written value (NA ÷ β) ^2. You can choose.

  Conventional reporter assays using the luciferase gene measure the amount of luminescence after lysing the cells, so only the expression level at a certain point in time can be captured, and the average value of the entire cell is measured. . Further, in measurement while culturing, changes in the expression level of cell colonies over time can be caught, but changes in the expression level in individual cells cannot be caught. In order to observe the luminescence of individual cells with a microscope, the amount of luminescence from living cells is extremely weak, so it can be exposed for a long time with a cooled CCD camera at the liquid nitrogen temperature level, or an image intensifier is attached. You have to do photon counting with a CCD camera. Therefore, the camera for detecting light emission is expensive and large. However, when observing the luminescence of individual cells exhibiting luciferase activity as a reporter gene product with a microscope, an image intensifier is attached if the apparatus for carrying out the luminescent sample imaging method according to the present invention is used. The quantitative image can be acquired using a cooled CCD camera at about 0 ° C. That is, if the apparatus for carrying out the luminescent sample imaging method according to the present invention is used, the luminescence of individual cells can be observed with a cooled CCD camera at about 0 ° C. in an alive state. No fire or photon counting device is required. That is, the luminescent sample can be imaged at low cost. In addition, if the apparatus for performing the luminescent sample imaging method according to the present invention is used, the luminescence of individual living cells can be observed over time while being cultured, and can also be observed in real time. . Moreover, if the apparatus for performing the luminescent sample imaging method according to the present invention is used, it is possible to monitor the response of drugs and stimuli under different conditions for the same cell.

  Here, in order to facilitate understanding of the luminescent sample imaging method, the luminescent cell imaging method and the objective lens according to the present invention, a conventional objective lens and luminescent image observation using the objective lens will be briefly described.

In general, the spatial resolution ε in microscopic observation is expressed by the following mathematical formula 1.
ε = 0.61 × λ ÷ NA (Formula 1)
(In Equation 1, λ is the wavelength of light and NA is the numerical aperture.)
Further, the diameter d of the observation range is expressed by the following mathematical formula 2.
d = D ÷ M (Formula 2)
(In Equation 2, D is the number of fields of view and M is the magnification. The number of fields of view is generally 22 to 26.)
Conventionally, the focal length of an objective lens for a microscope has been set to 45 mm according to international standards. Recently, an objective lens having a focal length of 60 mm has been used. If a lens with a large NA, that is, a high spatial resolution is designed on the assumption of this focal length, the working distance (WD) is generally about 0.5 mm, and even a long WD design is about 8 mm. When such an objective lens is used, the observation range is about 0.5 mm in diameter.

  However, when observing a cell group, tissue, or individual dispersed in a dish or glass bottom dish, the observation range may range from 1 to several centimeters. When it is desired to observe such a range with high resolution, the NA must be maintained at a large value while the magnification is low. In other words, since NA is the ratio of the lens radius to the focal length, an objective lens that can observe a wide range with a large NA needs to be low magnification. As a result, such an objective lens has a large aperture. In manufacturing a large-diameter objective lens, generally high accuracy is required in terms of uniformity of physical properties and coating uniformity of an optical material and lens shape.

In the case of microscopic observation, the light transmittance of the optical system, the numerical aperture of the objective lens, the projection magnification on the chip surface of the CCD camera, the performance of the CCD camera, etc. greatly affect the brightness of the image. The brightness of the image is evaluated by the square of the value obtained by dividing the numerical aperture (NA) by the projection magnification (β), that is, (NA / β) ² . Here, the objective lens generally has a relationship of the following Equation 3 between the incident aperture angle NA and the exit aperture angle NA ′, and NA ′ 2 indicates the brightness that reaches the observer's eyes, a CCD camera, or the like. Value.
NA ′ = NA ÷ β (Formula 3)
(In Equation 3, NA is the incident aperture angle (numerical aperture), NA ′ is the exit aperture angle, and β is the projection magnification.)
In a general objective lens, NA ′ is at most 0.04, and NA ′ 2 is 0.0016. Further, when the value of image brightness (NA / β) ² in an objective lens of a general microscope currently on the market was examined, it was in the range of 0.0005 to 0.002.

  However, using a microscope equipped with a commercially available objective lens as described above, for example, even if a cell expressing a luciferase gene and illuminating it is observed, the light emitted from the cell is visually observed. In addition, even if a light emission image taken using a CCD camera cooled to about 0 ° C. is observed, light emission from the cells cannot be confirmed. When observing a luminescent sample, it is not necessary to project excitation light necessary for fluorescence observation. For example, in epifluorescence observation, the objective lens satisfies both functions of an excitation light projection lens and a lens that collects fluorescence to form an image.

  Therefore, in order to observe light emission with a small amount of light with an image, an objective lens having large NA and small β characteristics is required. As a result, the objective lens tends to have a large aperture. Note that such an objective lens is required to be easily designed and manufactured by simplifying the function without considering the function of projecting the excitation light.

  In the research field using luminescence and fluorescence observation, time-lapse and moving image capturing are required to capture the dynamic functional expression of protein molecules in a sample. Recently, video observation of one molecule of protein using fluorescence has been performed. In these imaging operations, the exposure time per image frame becomes shorter as the number of imaging frames per unit time increases. Such observation requires a bright optical system, particularly a bright objective lens. However, since the amount of photoprotein is less than that of fluorescence, it often takes an exposure time of, for example, 20 minutes to image one frame. In order to perform time-lapse observation with such an exposure time, a dynamic change is limited to a very slow sample. For example, in cells that divide once every hour, changes within that cycle cannot be observed. Therefore, it is important to improve the brightness of the optical system in order to efficiently image a small amount of light while maintaining a high signal-to-noise ratio.

  The objective lens of the present invention manufactured based on the above circumstances has characteristics of a large NA and a small β as compared with the above-described objective lenses generally on the market. Therefore, NA′2 of the objective lens of the present invention is a large value. That is, it can be said that the objective lens of the present invention is a bright objective lens. Thereby, if a bright objective lens like the objective lens of this invention is used, the light emission from the light emission sample with little light quantity can be observed with an image. In order to observe a darker image, a CCD camera cooled to about 0 ° C. without mounting an image intensifier by mounting the objective lens of the present invention having a large numerical aperture on a stereomicroscope, Cell luminescence can be observed with images. In addition, there is a method of increasing the sensitivity with a CCD camera using liquid nitrogen cooling. In this case, the CCD camera becomes very expensive and large-scale. However, if the objective lens of the present invention is used, the light emission of the cells can be observed as an image even with a CCD camera using Peltier cooling. Moreover, the objective lens of the present invention has a large diameter of several to about 10 cm. Thereby, a moving luminescent sample that could not be an imaging target in the past or a luminescent sample distributed over a wide range can be set as an imaging target.

  The measurement principle for implementing the method of the present invention, the configuration of the optical system, and the like have already been described with reference to FIG. 1, but refer to the applications by the present applicant (Japanese Patent Application Nos. 2005-267531 and 2005-44737). May be. As described in the above-mentioned application, the applicant, through optical experiment, has a square value of (NA ÷ β) represented by a numerical aperture (NA) of the objective lens and a projection magnification (β) of 0. By taking an image with an optical system of 0.01 or more, proof data that can be imaged only by light emission generated from a single cell was obtained. Furthermore, the present applicant has proceeded with studies and found that the variation pattern of gene expression is different among a plurality of cells cultured in the same petri dish. Surprisingly, the above imaging conditions can also be applied to weak luminescent images handled in the application examples implemented in the present invention, and weak such as bioluminescence in a short time (for example, 1 to 20 minutes). Cell images can be captured with various luminescent components. Further, when the optical condition expressed by the square of the numerical aperture (NA) / projection magnification (β) is 0.071 or more, the objective lens of the imaging device can be imaged within 1 to 5 minutes. It was also found that cell images that can be analyzed can be provided.

  As shown in FIG. 1 described above, the apparatus for carrying out the imaging method according to the present invention is for imaging a sample 1 to be imaged with a short exposure time, and in real time, and has been conventionally employed. A high numerical aperture (NA) objective lens 2, a CCD camera 4 and a monitor 5 are basically configured. In the present invention, an apparatus having these basic configurations is referred to as a light emission microscope. In order to perform imaging in a dark field, the light emission microscope is preferably accommodated by a light shielding lid or housing as appropriate. Moreover, imaging can be automatically performed over a long period of time by combining a culture apparatus capable of maintaining appropriate culture conditions integrally with a light emission microscope. In addition, if it is a structure which has an imaging mechanism, it does not need to be in the form of a microscope, and may be in the form of a measuring instrument such as a microplate reader.

  Next, the configuration, form, and operation of the light emission microscope used in the present invention will be described. The basic feature of the luminescence microscope is that light emitted from a photoprotein expressed in cells in the microscope field can be monitored in a short time by imaging with a digital camera through an objective lens (NA 0.75) and an optical filter. That is. Since a gene expression pattern can be obtained in real time as a luminescence image, it is a technology that is expected to be applicable to a wide range of research subjects in gene expression assay systems using cells. The basic measurement system configuration is that the optical system adjacent to the culture apparatus unit is a sample observation unit, and the light emitted there is captured by a digital camera through an objective lens (NA 0.75, preferably NA 0.75 or more) and an optical filter. Later, data recording and analysis are performed on a digital image capturing personal computer. The culture apparatus part can be kept warm and moisturized by a heat plate and a chamber. The temperature in the culture apparatus is set to 25-37 ° C and the sample is observed. The sample is preferably observed at 37 ° C. The humidity is set to 0 to 100% and observed. Preferably, it is set to 60 to 100%. Furthermore, by acquiring a bright-field image in the same field as when optical luminescence imaging was performed, and superimposing the light-emission image and the bright-field image with image analysis software or the like, it is possible to identify the cells that emit light. .

  Definitions of terms relating to the method of the present invention are shown below.

<Promoter region of a gene whose expression is induced by stimulation>
Examples of the gene promoter region whose expression is induced by stimulation in the present invention include a promoter of an early gene. Examples of the promoter of the early gene used in the present invention include the c-fos promoter region. In addition, the promoter used in the present invention includes a counterpart of any animal species of the promoter. Here, the promoter region means an arbitrary region containing the minimum base sequence necessary for having promoter activity. For example, a part or all of a region of 500 to 2000 bases upstream from the transcription site of the gene can be used.

<Reporter gene>
The reporter gene in the present invention means a gene encoding a reporter protein that emits detectable fluorescence. For example, luciferase derived from firefly or Renilla can be mentioned. Furthermore, the gene which codes (beta) galactosidase and the gene which codes alkaline phosphatase can be mentioned, for example. As a substrate for causing the reporter gene to emit light, any substrate can be applied, and in particular, firefly luciferin and coelenterazine can be preferably applied.

<Optical imaging>
Optical imaging in the present invention means an imaging method for monitoring, recording and analyzing the presence, absence or intensity of a detectable signal emitted from a biological sample such as a cell or tissue. For example, to achieve optical imaging with a reporter gene in a cell into which the reporter gene has been introduced, the intensity of the signal emitted by the reporter gene is sufficiently sensitive so that the signal can be analyzed from outside the cell. There must be. Since optical imaging is readily applicable to automation, it can be used to monitor multiple gene expressions simultaneously. The technique for processing image information two-dimensionally or three-dimensionally at an arbitrary position of an imaged sample image is disclosed in Japanese Patent Application Nos. 2004-172156, 2004-178254, and 2004-342940 by the present applicant. Japanese Patent Application No. 2005-267531 may be referred to. The difference between time-series image acquisition in fluorescence observation and light emission observation is that there is no influence of extra light because light emission observation does not require optical scanning (laser scan) with excitation light. According to the imaging technique performed in the present invention, a luminescent image can be captured in real time.

B. Optical imaging method for gene expression <Production of gene expression vector>
When using animal cells, the expression vector preferably contains at least a promoter, a start codon, DNA encoding the protein of interest, and a stop codon. Also includes DNA encoding signal peptide, enhancer sequence, 5 'and 3' untranslated regions of gene encoding the protein, splicing junction, poly A addition signal, selectable marker region or replicable unit, etc. You may go out.

<Gene transfer method into cells>
Examples of methods for introducing genes into cells include calcium chloride method or calcium chloride / rubidium chloride method, lipofection method, electroporation method and the like. The gene-introduced cells used in the present invention include both transiently expressing cells and stable expressing cells.

<Optical imaging of gene expression by stimulation>
According to the present invention, it is possible to analyze the amount of luminescence in time series by a stimulus that contacts a cell with a substance capable of inducing any luminescent reaction. The cells to be stimulated and analyzed may be single cells or cell groups. By performing luminescence imaging, it becomes possible to trace any particular cell until what time, and the luminescence intensity or luminescence behavior due to the difference in cells can be accurately evaluated.

The number of cells into which the gene has been introduced (for example, 1 to 1 × 10 ⁹ cells, preferably 1 × 10 ³ to 1 × 10 ⁶ cells) can be used for a desired cell culture (for example, petri dish, multiple wells) In a desired nutrient medium (for example, D-MEM medium) using a multiplate or the like. A sample of this constant number of cells is preliminarily kept at a temperature optimum for the cells (for example, 25 to 37 ° C., preferably 35 to 37 ° C.) and water is injected to prevent the sample from being dried. A luminescent image is recorded by a digital camera through an objective lens in a sample observation section of the luminescence microscope that is installed in the culture apparatus. A substance (for example, a compound) for stimulating by contacting cells is added to the sample at a desired concentration (for example, 1 pM to 1 M, preferably 100 nM to 1 mM), and a desired time interval (for example, 5 minutes) is added. An image capable of image analysis can be prepared by recording a luminescent image in 5 hours, preferably 10 minutes to 1 hour. The image analysis of the prepared image can be automatically analyzed by analysis software that is commercially available or designed by a unique analysis algorithm. By displaying time-series parallel display or moving image on the display screen, analysis with the naked eye is also possible. As shown in the experimental results described later, in luminescence imaging of individual cells using the photoprotein of the present invention, a method for adjusting the length of exposure time is provided as an imaging condition. In particular, the imaging data obtained by dividing the exposure time into relatively short unit times (for example, 30 seconds or more and 5 minutes or less, preferably 30 seconds or more and 1 minute or less) are added according to the processing conditions of the sample, thereby providing a clear and high image. It has been found that both sensitive monitoring and / or analysis and long-term and accurate monitoring and / or analysis can be achieved. Therefore, the method of the present invention outputs the imaging data based on the exposure time divided into unit times according to the output conditions such as adding the number corresponding to the above processing conditions, or performs image processing. And / or provide a method of analysis. Here, the imaging data may be subjected to arithmetic processing such as averaging after the addition processing. On the other hand, the unit time may be set to be relatively long (for example, 10 minutes to 60 minutes, preferably 10 minutes to 30 minutes), and the imaging data may be divided according to the sample processing conditions. Thus, the method of the present invention is more advantageous by adjusting the length of the exposure unit time for obtaining one luminescent image, and preferably incorporating the step of dividing the image obtained in unit time. It becomes.

  Next, various conditions applicable to the method of the present invention will be discussed. First, in the present invention, a cell group as a sample or a living cell in a living tissue is treated with a luminescent component (for example, a luminescent protein fusion gene), and a substrate solution for inducing luminescence to the luminescent component is appropriately used. In the analysis based on the luminescence image of the cell by causing the cell to emit light by being present in the culture environment, the substrate concentration that does not impair the biological activity of the cell is set according to the culture period to be analyzed. It is a characteristic method. In order to perform image analysis over time while culturing living cells, it is preferable to appropriately set the concentration of the substrate solution for chemically exciting the luminescent component among various reagent conditions. I understood. Here, when the culture period is several days or more, by setting the concentration of the substrate solution to 700 μM or less, an effective luminescence image can be obtained without performing a luminescence reaction at an excessively high concentration. There was found. On the other hand, when the culture period is less than several days, the biological activity of the cells is maintained in a constant active state for several days to about one week by setting the substrate solution to a concentration of 800 μm or more and 1 mM or less. However, the luminescent image which can be analyzed can be provided stably. On the other hand, for concentrations higher than 1 mM, cells treated with a luciferin solution of 5 mM or less may be monitored or analyzed within a culture period of several days, but the toxicity to live cells increases as the culture period increases. Is done. Further, by setting the substrate concentration to 200 μM or more, there is an advantage that even a highly sensitive cell can observe a luminescent image while maintaining biological activity stably for a long period of time (for example, several weeks or more). is there. Here, typical examples of the substrate include firefly luciferin and coelenterazine, but it may be other types of substrates, chemical conversions of various substrates, or genetic engineering modifications. Coelenterazine can have a concentration range different from that of luciferin. For example, it is considered that 10 μM or more and 200 μM or less, particularly 50 μM or more and 100 μM or less is preferable. Coelenterazine solutions with concentrations greater than 100 μm may no longer be toxic to live cells and are suitable for limited use in culture periods of several days. When the culture period is several days or more, it is considered that an effective luminescence image can be obtained without performing a luminescence reaction at an excessively high concentration by setting the concentration of coelenterazine to 70 μM or less. When the culture period is less than a few days, the coelenterazine concentration is 80 μm or more and 100 μM or less, so that the biological activity of the cells is maintained in a constant active state for several days to about one week, It is considered that a luminescent image that can be analyzed can be provided stably. Thus, in the present invention, the substrate concentration is typically described as the luciferin concentration, but in the coelenterazine concentration, the concentration level is 1/10 of that. In the treatment method of the present invention, by continuously culturing living cells in the substrate concentration range as described above, the amount of luminescence that can always be imaged during the culture period is maintained while maintaining a state in which biological activity does not decrease. To keep.

  In another aspect, the present invention treats living cells with a luminescent component and causes the cells to emit light by allowing a substrate solution for inducing luminescence to the luminescent component to exist in an appropriate culture environment. In the analysis based on the luminescence image of the cell, the optical condition where the square of (NA ÷ β) expressed by the numerical aperture (NA) and the projection magnification (β) is 0.01 or more is measured from the cell. And a substrate concentration is set to 600 μM or less. As described above, the optical condition that the square value of (NA ÷ β) is 0.01 or more is an appropriate combination of the brightness and the projection magnification of the objective lens that can identify each cell emitting bioluminescence one by one. We ask for a match. When the square value of (NA ÷ β) as the optical condition is 0.039 or more, even if the substrate solution has a lower concentration (for example, 300 μM or less, preferably 100 to 200 μM), the image It is possible to always obtain a luminescent image that is clear enough to be analyzed.

  As a modified example of the present invention, the present invention can be applied to cells containing a fluorescent component (for example, green fluorescent protein (GFP), yellow fluorescent protein (YFP), etc.) in addition to the light emitting component. By applying both luminescence and fluorescence to the same cell, it is possible to perform comprehensive image analysis using the luminescence image and the fluorescence image, and facilitate detection of various substances inside and outside the cell. Here, the amount of excitation light is also determined according to the time required for acquiring the luminescent image (in the case of bioluminescence, the exposure time for an image sensor such as a CCD camera until an image that can be analyzed is obtained), for example, The longer the exposure time required for imaging, the lower the intensity of the excitation light for fluorescence can be set. As an example of good detection conditions, contact is made with a cell at a substrate concentration of 200 μM or more and 700 μM or less, and optical conditions are set so that a luminescent image is obtained within 30 minutes, preferably within 10 minutes. Since the cells can be kept in a biologically active state for a very long time (for example, several weeks or more), the activity change with the naked eye by various cell-based screening assays and images can be stably performed. On the other hand, if detection that depends only on high-sensitivity imaging such as fluorescence, infrared rays, and X-rays is performed, it is affected by continuous phototoxicity due to irradiation light to some extent. Can be a factor.

  As another aspect of the present invention, the test sample is a thick biological tissue (for example, a small fish such as a brain such as an upper visual nucleus, an embryo, a zebrafish or a small animal such as a mouse or a frog (especially Xenopus laevis)). When it is a part of an organ (eg, hand, foot, hair root, leaf, flower stem, root hair) of a plant (eg, Arabidopsis thaliana), the thickness suitable for luminescence imaging by bioluminescence is expanded two-dimensionally in a predetermined container. It is preferable to prepare a two-dimensional shape cell group (for example, a tissue slice section or a cell group film sheet), etc. In the study in the present invention, when performing luminescence imaging to the extent that individual cells can be identified, By using a living tissue having a thickness of an effective range of 150 μm or less, preferably 100 μm or less, particularly less than 85 μm, It is possible to individually image cells in the body tissue, because the cell group distributed in the body tissue often shows various reaction patterns (for example, gene expression level or expression level change pattern) It is possible to perform accurate analysis by grouping cells that show the same or similar reaction pattern from a large number of cell groups, or output a random reaction result at first glance. In this way, even in a thick biological tissue that is originally difficult to react or detect by weak light, it has a thin thickness within the effective range as described above. By arranging in a substantially constant state in dimension, analysis using weak light in a living tissue becomes possible.In the present invention, therefore, extremely weak such as bioluminescence is used. When performing detection to accurately know the dynamics of the whole living tissue by performing single-cell level analysis, the light signal due to weak light is imaged with a condensing lens of appropriate brightness (the objective lens in FIG. 1). Also included is a processing method to make the thickness as thin as can be formed.By setting various living tissues as thin as possible within the above numerical range, the cells do not overlap on the optical path for detection, so the image In addition, any biological sample prepared to the above-mentioned thickness has a thickness that is significantly thinner than before, so that the luminescent reaction with the substrate solution having the above-mentioned effective concentration is sufficient. It can proceed within an acceptable reaction time (approximately the same time as the luminescence reaction time in a single cell), and in addition to other drugs (eg, therapeutic agents (anticancer drugs, homeostasis) Rumon, etc.), diagnostics (biochemical test reagents, immunological test reagents, genetic test reagents, etc.), toxic test substances (genetic mutagens, carcinogens, allergens), preventive drugs (vaccines, etc.) In addition, it has a notable effect that it can guarantee sufficient reactivity with constitutional improvers (Chinese medicine, vitamins, supplements, etc.).

  On the other hand, when the biological tissue is prepared to a thickness of less than 30 μm, especially 20 μm or less, there are many cells damaged by the slicing process, and in image analysis, individual cell recognition is easily caused by analysis software. In some cases, the biological activity of the cells is reduced or inactivated in a short period of time (for example, less than 24 hours). Therefore, in the method of the present invention, when the test sample for obtaining the luminescent image is a living tissue, it is preferable to slice the sample into a thickness of 30 μm or more and 150 μm or less, or to form a sheet-like cell layer. . Preferably, by setting the thickness to 40 to 60 μm or more (particularly 50 μm or more), it is possible to prepare a wide variety of cells, particularly mammals (for example, humans and mice) on the detection optical path. In contrast, when a slide specimen such as a pathological section is stained with a high-intensity dye such as a fluorescent dye, a quantum dot, or a chemiluminescent dye, a light signal transmitted through the tissue using a section having a thickness of 300 μm or more is used. Since it is often imaged, it is extremely difficult to detect extremely weak luminescence such as bioluminescence from the outside of the sample. In the case of a biological sample having a thickness exceeding 100 μm, there are cases in which the six cells overlap each other. When the thickness is 200 μm or more, particularly 300 μm or more, it is very difficult to identify individual cells by the luminescence image. When these thicknesses are applied to living organisms such as animals, insects, and fish, early embryos, and biological materials for regenerative medicine, the depth of the imaging target is less than 300 μm, preferably less than 200 μm, particularly less than 100 μm. What is necessary is just to monitor and / or analyze the cell etc. which exist.

  As described above, in an example using a two-dimensional shape cell group derived from various living tissues, the present invention is applied when performing analysis of a living tissue by performing analysis of a single cell level with weak light, The weak light signal from the luminescence reaction without damaging the reactivity with the luminescent reagent for generating faint light in the cell or the reactivity with the drug that may cause a change in the luminescence reaction by the luminescent reagent in the cell. A method for analyzing living cells is provided, which uses a cell group that is two-dimensionally formed to have a substantially constant thickness so that can be detected from the outside.

[Experimental example]
In order to observe a luminescent sample (cell) using luciferase, it is necessary to administer luciferin as a luminescent substrate to the culture solution. Considering that luciferin is expensive and its effects on cells, it is desirable that it can be measured at a lower concentration. Therefore, optical imaging by luminescence was optimized as follows by setting the concentration of luciferin necessary for luminescence observation of living cells.

Bioluminescence after introducing a luminescent gene vector (Promega pGL3-control vector) into HeLa cells was optically imaged under the following conditions.
Imaging conditions: CCD camera DP-30 (manufactured by Olympus Corporation) with an operating temperature of 5 ° C., objective lens (oil immersion) with a projection magnification of 20 times and a numerical aperture of NA 0.8, imaging magnification of 5 times A luminescence image was obtained by exposing at room temperature for 1 minute using a luminescence microscope set to 1 (see FIG. 1).
Luciferin concentration: Under the above imaging conditions, each luminescent reaction can be achieved by adding the luciferin concentration to a final concentration of 1 mM, 0.5 mM, and 0.1 mM in the medium containing the luminescent gene-introduced cells. Carried out.

  As a result of this experimental example, it was found that a luciferin concentration in the range of 1 mM to 0.5 mM was sufficient. It was also found that the luminescence image was somewhat dark at 0.5 mM with respect to 1 mM luciferin, but no significant difference was observed (FIGS. 2 and 3). However, at 0.1 mM, the luminescent image becomes considerably darker after 1 minute exposure. In this case, by changing the exposure to 5 minutes, a high-resolution luminescent image corresponding to exposure at 0.5 mM for 5 minutes. I was able to get. In addition, in the case of 1-minute exposure, it is a raw screen (FIGS. 2 to 4) without drawing a dark screen. FIG. 2 shows results at 1 mM, FIG. 3 shows 0.5 mM, and FIG. 4 shows results at each luciferin concentration of 0.1 mM. It is. In the case of exposure for 5 minutes (0.1 mM luciferin, exposure for 5 minutes), the screen is obtained by subtracting the dark screen (FIG. 5-1).

  Next, the average value of the luminescence intensity of 10 identical cells was calculated under each condition, and the values were normalized and compared with 0.1 mM luciferin having the weakest luminescence intensity at 1 minute exposure. The comparison results are shown in the graph of FIG. In the case of 1 minute exposure, the amount of luminescence does not change so much at luciferin concentrations of 1 mM and 0.5 mM, but at 0.1 mM, it decreases by 1/4 to 1/3. However, even with 0.1 mM, if the exposure is extended to 5 minutes, 1 mM luciferin reaches the same amount of luminescence as 1 minute exposure.

  Note that the present invention is not limited to the description given in the above-described embodiments and experimental examples, and can include various aspects and equivalents thereof. Further, the various processing conditions described in the present invention may be arbitrarily combined. Furthermore, it may be in the form of a product that is manufactured by performing the treatment method of the present invention and is stabilized so as to be distributed (for example, drying treatment, vacuum packaging, sheet shaping treatment, thickening saccharide solution impregnation treatment, etc.). . In addition, in the case of a two-dimensional shape cell group in which a living tissue is thinned, a bottom surface of a container such as a single chip substrate or a single petri dish may be covered with a necessary surface area, or many different living tissues Each of the two-dimensional shape cell groups obtained by subdividing can be microarrayed by spotting in a matrix at different address positions.

  For details of the optical conditions regarding the apparatus according to the above description, refer to Japanese Patent Application Nos. 2005-267531, 2005-44737, and 2006-61321 by the applicant, and the optical conditions including the objective lens and the imaging lens are as follows. Can understand. For image information processing and application examples after image information acquisition related to the present invention, image cytometers described in JP-A-11-271209, JP-A-2000-279168, JP-A-10-293094, and JP-A-3-255365 are described. The configuration relating to this can be referred to for the present invention. However, since conventional image cytometers use samples that generate optical signals that can be observed with the naked eye, such as fluorescence, the sample or measurement beam is moved in sequence as in a flow cytometer or a laser scanning microscope. In contrast to the method and apparatus for analyzing individual cells, the method and apparatus of the present invention is capable of imaging faint light (eg, bioluminescence) invisible to the naked eye on a plurality of cells simultaneously. It is greatly different in that it is capable of comprehensive analysis, that is, comprehensive analysis of all cells in the field of view or in a predetermined container at the same time in that an accumulation type photodetection device is provided. The above-described effects can be achieved by executing the imaging conditions of the conventional apparatus as described above by an appropriate control unit that is adjusted according to the imaging conditions according to the sample processing conditions according to the present invention. Become.

  When measuring a luminescence phenomenon caused by a bioluminescent protein, the luminescence intensity is still very weak at the time of sample setting, and almost cannot be detected as a light signal by a light receiver. Therefore, the internal structure of the cell cannot usually be observed. That is, the objective lens cannot be focused while confirming the cell that is the observation target in the sample, as in normal microscope observation. Therefore, as a bright field image observation, light from a light source such as a halogen lamp is projected onto the test sample through the illumination optical system to obtain a bright field image of the microscope by the test sample, and based on this, the focus position of the objective lens is determined. decide. That is, in the bright field observation, two substantially central positions on the optical axis of the objective lens that can obtain a high contrast image are set as the focus positions of the objective lens. Thereby, when the luminescence intensity by the bioluminescent protein becomes large, a clear luminescent image focused on the CCD camera can be obtained. The objective lens can be appropriately oil-immersed according to the desired magnification. Further, which magnification is selected may be appropriately selected according to the size of the sample to be evaluated (or analyzed). In the examination with the illustrated apparatus (light emission microscope), the optical system can be designed so that the emission image can be obtained at 40 to 100 times.

  The present invention is applicable to various modes as shown below.

[Aspect 1]; Analysis Method and Reagent Kit Using RNA Interference This aspect is a method for imaging information related to various living bodies and visually interacting substances in biological samples such as cells. The present invention relates to an analysis method and reagent kit for detecting an action. More specifically, the present invention relates to a method applied to a reporter assay that quantitatively detects intracellular interactions using bioluminescence. As an example, the present invention includes an analysis method using RNA interference and a reagent kit.

  The technology for examining living organisms from inside and outside has been remarkably developed. Every living body on the earth has living body-related information that can be directly and uniquely applied to medical results such as diagnosis, treatment, and preventive medicine as promising biological uses. As an example of biological information, there is a case of obtaining a biological interaction (hereinafter abbreviated as an interaction) obtained from a cell (or a biological material containing the cell) living in a living body or in vitro. . Many techniques for examining interactions in living cells are realized by methods and reagent kits that detect reporter substances that generate optical signals from the outside of the cell. As such a reporter substance, for example, a method of applying a green fluorescent protein (GFP) derived from Aequorea jellyfish to a living cell and performing image analysis is known. In particular, RNA interference (RNAi) is a gene function analysis method using a powerful reporter assay that has rapidly emerged in recent years, and is encoded by degrading specific target mRNAs in cells and in vivo. This is a method for examining the function of a gene by knocking out or knocking down the expression of a protein. RNA interference is effective in a sufficient time (2 to 72 hours) for siRNA to exert a biological effect after siRNA introduction, but depending on the target and experimental conditions, the degree of RNA interference effect, RNA interference Time to effect is different. Conventionally, cells at certain time points have been prepared and confirmed by techniques such as Northern analysis, RT-PCR, and immunolabeling (see JP2003-219893).

  However, in general, in the conventional method, in order to examine the degree of the siRNA interference effect and the time until the RNA interference effect 8 appears, it is necessary to prepare a large number of biological samples, and the measurement interval is also 3 to 6 hours. In many cases, the condition setting is rough. In addition, the efficiency of siRNA transfection varies depending on the nature of the cell, the period of the cell cycle, the cell culture density, and the material to be transfected. Therefore, the averaged measurement data may not accurately reflect individual results.

  Therefore, this aspect 1 aims to provide a method and a reagent kit that can accurately analyze the results of RNA interference performed inside and outside individual cells. Another object of the present invention is to provide a method and a reagent kit that can easily analyze a gene expression level over a long period of time. In this embodiment 1, a luminescence image of a cell into which a luciferase gene has been introduced and a luminescence image due to RNA interference are taken with an optical system that satisfies the following conditions. According to the optical experiment by the present applicant, imaging is performed with an optical system in which the square of (NA ÷ β) expressed by the numerical aperture (NA) of the objective lens and the projection magnification (β) is 0.01 or more. Thus, it was proved that an image can be formed only by luminescence generated from a single cell (see Japanese Patent Application No. 2005-267531). Furthermore, the present applicant has proceeded with studies and discovered that the variation pattern of gene expression is different among a plurality of cells cultured in the same petri dish (see Japanese Patent Application No. 2005-44737). Surprisingly, the above imaging conditions can also be applied to a weak luminescence image in RNA interference, and a cell image with a weak luminescence component such as bioluminescence can be captured in a short time (eg, 1 to 20 minutes). Further, according to the optical condition investigated by the inventor, when the optical condition expressed by the square of the numerical aperture (NA) / projection magnification (β) of the objective lens of the imaging apparatus is 0.071 or more. It was found that a cell image that can be imaged within 1 to 5 minutes and can be analyzed is provided.

  Therefore, the analysis method of aspect 1 is a method of analyzing interaction between cells inside and outside based on image information, wherein a luminescent substance as a biological excitation substance is labeled on at least one of the substances that can interact. Including the step of causing an interacting substance to be present in one or more cells, optically imaging the resulting luminescence, and imaging the captured optical information for a single cell, wherein the image representation interacts It is characterized by the presence and / or amount of weak light due to. Here, this aspect 1 preferably includes living cells as the biological sample. In addition, this embodiment 1 preferably contains a component that brings about bioluminescence as a biological excitation substance. This embodiment 1 can be made into a reagent kit, and includes a component capable of inducing an interaction and a substrate component capable of inducing luminescence of the luminescent substance. In addition, this aspect 1 preferably includes a step of evaluating an interaction with a drug that affects the activity of the biologically-excited substance in any of the methods described above.

  In this mode 1, unless otherwise specified, “luminescence” means bioluminescence, and “luminescence material” has any luminescence that can give excitation energy to a fluorescent material in BRET. Means a substance.

  According to this aspect 1, more accurate data can be obtained by performing continuous data acquisition for only transfected cells by performing RNA interference with a reporter assay using luminescence. Further, since it is possible to observe the gene expression level over a long period of time, it can be widely used for clinical uses such as drug discovery research.

<RNAi preliminary experiment>
In order to perform RNA interference experiment evaluation regarding this embodiment 1, first, as an RNAi preliminary experiment, an inhibition experiment by siRNA against pGL3 gene was attempted using HEK293 cells. This time, it was introduced by Lipofectamine2000. An observation image by the luminescence imaging system LV200 (Olympus Corporation) is shown in FIG. 6-1 (Ixon EM-CCD camera (Andor), EMgain1000, exposure time 10 min). Regarding the dish to which siRNA was added, there were few cells expressing the GL3 gene, and the expression of the GL3 gene seemed to be suppressed (right-side observation image in FIG. 6A). Further, numerical data obtained by the luminometer Kronos (Ato) is shown as a graph in FIG. 6-2. Measurements by Kronos, 10 ⁶ cells / ml in HEK293 cells to pGL3 Control vector, after introducing processing siRNA, was achieved through addition of luciferin 100μM processed cells from 10 ^{5 /} ml after ¹⁰⁶ / ML24 hours It was. When HEK293 was used, there was a difference in the sticking of cells between plastic and glass bottom, so we compared two dishes, but both showed suppression of expression by siRNA.

<RNAi experiment>
Next, in order to evaluate RNA interference experiments, the degree of RNA interference was confirmed using siRNA against the fluorescent protein GFP. A pEGFP-C1 Vector (BD Clontech) was introduced into HeLa cells, and GFP-expressing cells were transfected with siRNA for causing RNA interference. When the cells were observed with a fluorescence microscope IX81 (Olympus) after the transfection, the expression level of GFP in the cells was reduced as compared with the cells without siRNA introduction. In order to detect RNA interference using luciferase as a reporter gene, the GFP gene was introduced into the multicloning site of psiCHECK ^™ -92 Vectors (Promega Corporation) to prepare a construct. When a vector such as psiCHECK ^™ -2 Vector is used, when the RNA interference mechanism by the synthetic siRNA for the target gene is started, the renilla luciferase gene connected to the target gene is also cleaved and decomposed. Activity is reduced. In this example, it was attempted to easily monitor the RNA interference effect by continuously imaging such a decrease in activity under a microscope.

After introducing the construct prepared into HeLa cells, transfection of siRNA against GFP was performed under the conditions where the effect of siRNA was confirmed, and observation was performed with a luminescence microscope. After adding EnduRen ™ Live Cell Substrate (Promega Corporation) to cells in a 35mm glass bottom dish, introduce CO ₂ gas into the incubator of the luminescence microscope and observe for 3 days while culturing at 37 ° C. went. The results of observation over time with a light emission microscope are shown in FIG. 6-3 as a graph. When a comparison was made between HeLa cells into which siRNA was introduced and HeLa cells into which no siRNA had been introduced as a negative control, only the HeLa cell line into which siRNA had been introduced showed a decrease in luminescence. RNA interference to GFP was detectable as a decrease in luminescence from Renilla luciferase.

  As mentioned above, although this aspect 1 was demonstrated by the example which image-analyzes the RNA interference in a living cell, it is inherent in description of the aspect mentioned above, and by all inventions etc. which were filed by others (or other companies) before this application The invention includes technical ideas belonging to all categories that are not obvious, and has the scope of right to cover the equivalents covered by the technical ideas or the inventions of all subordinate concepts not disclosed in the present invention.

  In addition, this aspect 1 directly and uniquely includes various modified examples derived from the following consideration. This is because various modifications are merely diverted to various applications through the considerations common to the present invention.

[Discussion regarding aspect 1]
RNA interference is a typical technique that steadily reflects the presence or absence of protein interactions inside and outside the body. If it is attempted to detect the interaction only by the light intensity, the reliability is lowered due to a non-specific reaction, a noise signal such as a contaminant or autofluorescence. In general, fluorescence is excited by irradiation energy of excitation light emitted from the outside. Conventional imaging methods relating to a living body utilize excessive irradiation energy that is visualized so as to be visible as much as possible. Compared with fluorescence, light emission does not require photoexcitation, so there is no phototoxicity, and the operation is simple and easy to implement. In addition, the intensity of fluorescence is unstable, but the emission is stable. Bioluminescence is suitable for evaluation of changes (for example, monitoring of gene expression fluctuations) and continuous, wide-field light reception as a (non-obvious) viewpoint that cannot be imagined from fluorescence detection with excessive energy by excitation light irradiation. . In other words (upgrading), it is difficult to execute an inspection item in which the light intensity changes in accordance with a change in intracellular activity based on image information based on excessive light energy. In addition, as a viewpoint that cannot be conceived from the detection of bioluminescence by spectroscopic measurement or photon counting (non-obvious), comparing multiple detection targets in the same field of view or the same sample individually, and evaluating individual changes It is. In the first place, the bioluminescence is too dark to be observed with the naked eye or a microscope eyepiece. As with conventional microscopes, when images are too dark to acquire an image, it is necessary to take out cells one by one or move them to separate containers for inspection, which requires much time and man-hours. . Furthermore, it is troublesome to perform a procedure of lysing or crushing cells one by one for the inspection inside the cells. Using separate containers or individual procedures is unsuitable for continuous or multiple examination of living cells or tissues. By applying the method of the present invention, it is possible to examine a plurality of changes such as gene expression individually for each sample of cells or the like individually. In addition, there is the appeal that data can be taken from the same cell as many times as possible. When these considerations are combined, the novelty and inventive step of the present invention will be easily understood.

[Aspect 2]; Method and apparatus for imaging fluorescence emitted by fluorescent molecules excited by energy transferred by bioluminescence resonance energy transfer (BRET) using fluorescence as an indicator This aspect 2 relates to various living bodies The present invention relates to a method for imaging information, and a method and apparatus for visually detecting an interaction between substances that can interact in a biological sample such as a cell. Specifically, this embodiment 2 relates to a method applied to a reporter assay for quantitatively detecting intracellular interactions using bioluminescence. As an example, this embodiment 2 is a fluorescent molecule excited by energy transferred by bioluminescence resonance energy transfer (BRET) using fluorescence as an index when light energy by intracellular luminescence moves to a nearby fluorescent molecule. The present invention provides a method and an apparatus for imaging fluorescence emitted from a light source.

  As another example of biological information, by cloning a gene constituting a protein having a function of synthesizing a substance capable of generating an optical signal such as GFP as a reporter substance, and further performing genetic engineering modification, Variants have been made that have desired optical and biological properties such as brightness, absorption / fluorescence wavelength, pH sensitivity and the like. A specific region in the cell can be visualized by adding a signal indicating localization in the cell to these fluorescent protein genes, and introducing and expressing the signal in the cell. In addition, by fusing with a functional protein, the localization and behavior of the protein in the cell can be observed. Furthermore, if the interacting proteins are labeled with fluorescent proteins of different colors, when the proteins approach each other due to the interaction, the energy of one fluorescence excites the other fluorescent molecule, and the fluorescence shifted to a longer wavelength is emitted. Observed (FRET). By visualizing this wavelength shift, it is possible to detect intermolecular interactions in the cell (Otuji et. Al., Analytical Biochemistry. 329 (2004) 230-237, Monitoring for dynamic biomolecules molecular biological processing. transfer system using secreted luciferase).

  However, in order to perform FRET measurement, excitation light must be applied to the cells. In long-term measurements such as tens of hours or days, cells are damaged by the phototoxicity of excitation light. On the other hand, there is a method for measuring intermolecular interactions using bioluminescence resonance energy transfer (BRET) in which a fluorescent protein serving as an energy donor is replaced with a photoprotein such as luciferase without using excitation light. The fluorescent protein that becomes the acceptor is excited by the light energy generated by light emission, and the ratio of the light intensity between the emission wavelength and the fluorescence wavelength is measured (Otsuji et al, 2004). In this case, there is no need to irradiate the cells with excitation light, so there is no phototoxic effect. However, since the luminescence intensity in living cells is extremely weak, the fluorescence excited by the energy transferred from this luminescence is also weak, and it is expected that the light intensity is approximately half that of luminescence. Therefore, the conventional CCD camera or photomultiplier tube scanning method has no ability to image fluorescence from the acceptor due to cell emission and BRET.

  Therefore, this aspect 2 aims to detect any interaction in a biological sample directly and minimally invasively. In addition, this aspect 2 is intended to provide a method and an apparatus for imaging image information caused by a change in a weak light signal such as BRET in units of cells.

[Means for solving problems]
The luminescence image of the cell into which the luciferase gene is introduced and the fluorescence image by BRET are imaged with an optical system that satisfies the following conditions. According to the inventor's optical experiment, imaging is performed with an optical system in which the square of (NA ÷ β) expressed by the numerical aperture (NA) of the objective lens and the projection magnification (β) is 0.01 or more. Thus, it was proved that imaging can be performed only by luminescence generated from a single cell (see Japanese Patent Application No. 2005-267531). Surprisingly, this imaging condition can be applied to a weak fluorescent image in BRET, and a cell image with a weak luminescent component such as bioluminescence can be captured in a short time (for example, 1 to 20 minutes). Further, according to the optical condition investigated by the inventor, when the optical condition expressed by the square of the numerical aperture (NA) / projection magnification (β) of the objective lens of the imaging apparatus is 0.071 or more. The present inventors have found that a cell image that can be imaged within 1 to 5 minutes and can be analyzed can be provided.

  Therefore, the method of this aspect 2 is an imaging method for acquiring biologically relevant information as image information. Biology that causes an optically detectable change by biological energy to an imaging target to be imaged. An image-related information corresponding to an output provided by the biological energy is preferentially acquired from the imaging target by causing an activator to act.

  In addition, in the method of this aspect 2, in the method of capturing image information relating to intracellular interaction, a luminescent substance and a fluorescent substance as biologically exciting substances are labeled on both of the substances that can interact with each other. A step of causing an interaction substance to exist in one or more cells, optically imaging energy transfer from a luminescent substance to a fluorescent substance as a result of the interaction, and imaging the optical information thus captured for a single cell And the image representation indicates the presence and / or amount of weak light resulting from the interaction.

  Here, it is preferable that the biological sample contains living cells.

  In addition, in the imaging apparatus for biological information, the apparatus of aspect 2 brings about a means for aiming at an imaging target to be imaged and a change that can be detected optically by biological energy with respect to the imaging target. Image information acquisition means for preferentially acquiring output that can be provided by the biological energy as image information in a state where a biological excitation substance is allowed to act.

  Also, this aspect 2 includes a program for automatically executing the above method. Further, this aspect 2 includes software in which the above program is written so as to be readable. In addition, this aspect 2 includes an analysis method for analyzing living body related information based on the image information acquired by the above method. The analysis method described above preferably includes a component that causes bioluminescence as a biological excitation substance. Examples of the luminescent component include a luminescent substrate and a luminescent protein fused with a luminescent enzyme or a gene encoding a luminescent enzyme. If the above method further includes the step of evaluating the interaction with the drug that affects the activity of the biologically-excited substance, it contributes to medical research, drug discovery research, drug efficacy, toxicity The evaluation can be performed under the same conditions as the investigation of living organisms. In addition, this aspect 2 includes a reagent kit having optical characteristics suitable for the image information acquisition means in the above apparatus.

  In this aspect 2, unless otherwise specified, “luminescence” means bioluminescence, and “luminescent material” has any light emission that can give excitation energy to a fluorescent material in BRET. Means a substance.

  According to this aspect 2, FRET using a fluorescent protein is used for measurement of intermolecular interactions in cells. In this method, since it is necessary to irradiate cells with excitation light, long-time light irradiation damages the cells. By using a BRET system in which the fluorescent protein on the energy donor side in FRET is replaced with a photoprotein such as luciferase, the influence of light irradiation on the cells can be eliminated. Therefore, long-term observation over several days to several weeks or more is possible, and by imaging this over time, the intermolecular interaction in the cell can be displayed as an image.

  With respect to this embodiment 2, an example in which apoptosis is detected by BRET using luciferase and a fluorescent protein will be described below. That is, the activity of caspase 3 activated by induction of apoptosis is detected by luminescence resonance energy transfer (BRET). The donor photoprotein gene is Gaussia Luc (Lux Biotechnology) and the acceptor YFP gene is pYFP (Clontech), which is activated between luciferase and YFP during apoptosis induction. A vector in which a recognition sequence of caspase 3 (DEVD; amino acid temporary notation, aspartic acid, glutamic acid, valine) is introduced is prepared. An amino acid such as glycine (G) selenium (S) is inserted as a linker between the luciferase and DEVD sequences and between DEVD and GFP.

  In this state, FRET occurs in the energy of luminescence by luciferase, which moves to the adjacent YFP and emits fluorescence (529 nm) from YFP (BRET). When apoptosis is induced and caspase is activated, luciferase-YFP molecules are digested and BRET is eliminated, so that luminescence by luciferase (480 nm) is observed.

[Preparation of expression vector in E. coli]
PRSET_B (Invitrogen), an expression vector in E. coli, is digested with restriction enzymes BamHI and EcoRI and purified. In addition, the Gaussia luciferase gene and YFP are amplified by PCR. The PCR primer on the forward side of the YFP gene contains a recognition sequence for the restriction enzyme BamHI at 5 ', and the primer on the reverse side contains a caspase recognition sequence and a recognition sequence for the restriction enzyme KpnI on the 3' side. The primer on the luciferase gene Forward side contains a KpnI recognition sequence at 5 ′, and the primer on the reverse side contains an EcoRI recognition sequence at 3 ′. Each gene is amplified and purified by PCR, and then digested and purified by restriction enzymes BamHI and KpnI and KpnI and EcoRI. Next, the pRSET vector, luciferase gene, and YFP gene digested with restriction enzymes are ligated with DNA ligase to construct a YFP-luciferase-vector as shown in FIG.

[Measurement with purified protein]
The vector of FIG. 7 was introduced into E. coli (JM109 (DE3)) and cultured overnight, then E. coli was disrupted using a French press, and the produced YFP-luciferase protein was affinity purified on a nickel-agarose gel (Ni- NTA Superflow, Qiagen), and the emission spectrum was measured over time under the following conditions (Lumiful Spectrocapture, Ato).

  FIG. 7-2 shows the change over time in the emission intensity at each wavelength under the following conditions. Treatment with 50 μg / ml YFP-4-DEVD-6-Gaussia, 60 μM Coelenterazine, 2 units Caspase 3 in Tris-HCl buffer (pH 8.0) Caspase 3 was carried out at 37 ° C. Since BRET occurs before the reaction, YFP fluorescence (546 nm) is strong, but when the reaction proceeds and the DEVD site is cleaved, BRET is eliminated, YFP fluorescence decreases, and Gaussia emission (478 nm) increases. .

  FIG. 7-3 shows the change with time of the emission spectrum from 0 minutes to 180 minutes under the following conditions. 0.1 M Tris-HCl buffer (pH 8.0), 50 μg / ml purified protein, 60 μM coelenterazine, 2 Unit caspase 3, 37 ° C. Observing changes in YFP fluorescence (546nm) and Gaussia emission (478nm) over time, BRET occurs before the reaction, so YFP fluorescence (546nm) is strong, but when the reaction proceeds and the DEVD site is cleaved BRET is eliminated, YFP fluorescence decreases, and Gaussia emission (478 nm) increases.

  FIG. 7-4 is a graph showing the ratio of the intensity at 480 nm to the intensity at 529 nm. Thus, it is possible to measure how the YFP-luciferase fusion protein is digested by Caspase 3 and BRET is eliminated.

[Preparation of expression vector in mammalian cells]
The fusion protein gene site prepared in FIG. 7-1 is replaced with pcDNA3.1, which is an expression vector in mammalian cells. The prepared vector pcDNA3.1 is the same as the vector pRSETB shown in FIG. 7-1 except that the type of vector is different.

[Gene transfer into cells and preparation for measurement]
A luciferase-GFP vector prepared by the lipofectin method is introduced into HeLa cells cultured in D-MEM. The culture medium is discarded the next day, and the cells are washed with Hank's balanced salt solution (HBSS) and replaced with 1 ml of HBSS. Coelenterazine (Promega), a luminescent substrate, is added to this at a final concentration of 60 μM and incubated at 37 ° C. for 1 hour.

[Measurement]
In order to induce apoptosis, 1 μl of anti-Fas antibody (Medical and Biological Laboratories) and cycloheximide (final concentration 10 μM) were added to start the measurement. First, shoot without filter for 5 minutes, then shoot for 5 minutes through a 510 nm long pass filter. This is taken for 6 hours at 30 minute intervals. The amount of fluorescence of GFP was a value after filtering at 510 nm, and the amount of luminescence by luciferase was a value obtained by subtracting the amount of light after filtering from the amount of light without filter. FIG. 8A is an image without a filter before the start of stimulation. FIG. 8-2 schematically shows an image with and without a filter 3 hours after the start of stimulation. FIG. 9 shows the change in luminescence of luciferase and the fluorescence intensity of YFP over time in the square region. At first, the fluorescence amount of YFP is strong due to BRET, but it can be seen that the amount of luminescence by luciferase increases when the luciferase-YFP complex is digested by caspase and BRET is eliminated. Thus, by monitoring the ratio of the luminescence intensity of luciferase and the fluorescence intensity of YFP, the activity of caspase, that is, the degree of apoptosis can be detected.

  As described above, this aspect 2 has been explained by an example of image analysis of BRET of a living cell. However, this aspect 2 is inherent in the above-described embodiment and examples, and has been filed by another person (or other company) prior to this application. It includes technical ideas belonging to all categories that are not obvious even by any equivalent invention, and includes the equivalents covered by the technical ideas or the scope of rights encompassing the inventions of all subordinate concepts not disclosed in this embodiment 2. Have.

  In addition, this aspect 2 directly and uniquely includes various modified examples derived from the following consideration. This is because various modifications are merely diverted to various applications through the same considerations as those in the second aspect.

[Consideration regarding aspect 2]
BRET and FRET are typical techniques that steadily reflect the presence or absence of protein interactions inside and outside the body. If it is attempted to detect the interaction only by the light intensity, the reliability is lowered due to a non-specific reaction, a noise signal such as a contaminant or autofluorescence. In general, fluorescence is excited by irradiation energy of excitation light emitted from the outside. Conventional imaging methods relating to a living body utilize excessive irradiation energy that is visualized so as to be visible as much as possible. Compared with FRET, BRET does not require excitation and is easy to experiment. In addition, the intensity of fluorescence is unstable, but the emission is stable. Bioluminescence or accompanying fluorescence (for example, BRET) can be evaluated (for example, monitoring variation in gene expression) or continuously, as a (non-obvious) viewpoint that cannot be conceived from fluorescence detection by excessive energy due to excitation light irradiation. It is also suitable for receiving a wide field of view. In other words (upgrading), it is difficult to execute an inspection item in which the light intensity changes in accordance with a change in intracellular activity based on image information based on excessive light energy. In addition, as a viewpoint that cannot be conceived from the detection of bioluminescence by spectroscopic measurement or photon counting (non-obvious), comparing multiple detection targets in the same field of view or the same sample individually, and evaluating individual changes It is. In the first place, bioluminescence, and also bioluminescence-derived fluorescence excited by light energy of bioluminescence (for example, BRET) is too dark to be observed with the naked eye or a microscope eyepiece. As with conventional microscopes, when images are too dark to acquire an image, it is necessary to take out cells one by one or move them to separate containers for inspection, which requires much time and man-hours. . Furthermore, it is troublesome to perform a procedure of lysing or crushing cells one by one for the inspection inside the cells. Using separate containers or individual procedures is unsuitable for continuous or multiple examination of living cells or tissues. By applying the method of this aspect 2, it is possible to examine a plurality of changes in gene expression and the like individually for each sample of cells or the like. In addition, there is the appeal that data can be taken from the same cell as many times as possible. By summing up these considerations, the novelty and inventive step of the above aspect 2 will be easily understood.

[Aspect 3] Method for analyzing gene expression level change in a single cell [Technical Field]
This aspect 3 relates to a method for analyzing a change in the expression level of a gene in a single cell by optically imaging a signal emitted by a reporter gene in the present invention.

[Background technology]
As a conventional method for analyzing gene expression by optical imaging, a fluorescent protein is used as a reporter gene (see Japanese Translation of PCT International Publication No. 2004-5000576), and downstream of the promoter region of the gene whose expression level is to be observed. A gene expression vector in which green fluorescent protein (GFP), blue fluorescent protein (BFP), and red fluorescent protein (RFP) genes are linked to each other is prepared and introduced into cells, and excitation light that matches each fluorescent protein is generated. The amount of fluorescence obtained by irradiation is measured. Since the amount of fluorescence is proportional to the expression level of the gene, the expression level of the target gene can be observed. On the other hand, as a gene expression analysis method using bioluminescence, as disclosed in Japanese Patent Application Laid-Open Nos. 2005-1118050, 2000-354500, and Japanese Patent No. 3643288, a gene whose expression is induced by stimulation via G protein. A gene expression vector in which a luciferase gene is linked downstream of the promoter region is prepared and introduced into the cell, and light emitted from the cell after stimulation via G protein is detected with a luminometer. Since the amount of luminescence is proportional to the expression level of the gene, it is possible to observe the expression level of the gene whose expression is induced by stimulation.

[Problems arising from the above background]
In the prior art using a fluorescent protein, phototoxicity to cells due to excitation light irradiation is not considered, and it is not possible to cope with the point of maintaining cells in a state where they can continue to survive. Therefore, paying attention to this point, it is an object to provide a method for analyzing gene expression in a single cell using bioluminescence, in which there is no need to irradiate excitation light, and thus there is little damage to cells. In addition, the conventional technology using fluorescent proteins does not consider the background of excitation light itself or autofluorescence of intracellular substances, and quantitatively determines the amount of signal generated by the reporter gene with a high S / N ratio. I cannot cope with the point of doing. Therefore, it is also an object to provide a method for analyzing gene expression in a single cell using bioluminescence, which uses a photoprotein that does not require excitation light and has a relatively low background. Furthermore, in the prior art using a photoprotein, it is not considered to observe a signal generated by a reporter gene in a single cell, and it is possible to cope with optical imaging of a detectable signal generated by a reporter gene. I can't. Therefore, focusing on this point, it is also an object to provide a method for analyzing gene expression in a single cell using bioluminescence, in which imaging is performed with a luminescence microscope so that cells emitting a signal generated by a reporter gene can be identified. Become.

[Means and effects for solving the above problems]
In order to achieve the object of solving the above-mentioned problem, as described later, a luciferase gene such as firefly or Renilla is used as a reporter gene, so that it is not necessary to irradiate cells with excitation light to generate a signal. It has been found that optical imaging can be performed in a state in which the background due to the autofluorescence of the intracellular substance is reduced and the injury of the cell is reduced. In addition, in a method of analyzing gene expression level in cells using a luciferase gene as a reporter gene, when observation is performed using a luminescence microscope, expression of the reporter gene can be optically imaged in a single cell by adding a ligand. I found out. In addition, by using the promoter region of the human c-fos gene as a promoter to control the expression of the reporter gene, it was found that when ATP or serum was added as a ligand, increased expression of the reporter gene product could be optically imaged in a single cell. . Furthermore, by capturing bright-field images of cells in the same field of view as the luminescent image, overlaying the images, and identifying the cell from which the luminescent image was obtained, the change in gene expression over time in a single cell is obtained. As a result, the invention according to the third aspect has been completed.

[A. Definition of word in aspect 3]
<Promoter region of a gene whose expression is induced by stimulation>
Examples of the promoter region in Aspect 3 include a promoter of an early gene. Examples of the promoter of the early gene used in Aspect 3 include the c-fos promoter region. In addition, the promoter used in aspect 3 includes the counterpart of any animal species of the promoter. Here, the promoter region means an arbitrary region containing the minimum base sequence necessary for having promoter activity. For example, a part or all of the region of 500 to 2000 bases upstream from the transcription site of the gene can be used.

<Reporter gene>
The reporter gene in aspect 3 means a gene encoding a reporter protein that emits detectable fluorescence. For example, luciferase derived from firefly or Renilla can be mentioned. Furthermore, the gene which codes (beta) galactosidase and the gene which codes alkaline phosphatase can be mentioned, for example.

<Substance>
The “substance” used in the invention described below means a natural substance existing in nature or any substance prepared artificially. Specifically, for example, any compound synthesized chemically can be mentioned. The type and molecular weight of the compound are not particularly limited. When the substance is a protein or peptide, those isolated from living tissues and cells and those prepared by genetic recombination or chemical synthesis are also included. Furthermore, those chemical modifications are also included.

<Ligand>
The ligand of the G protein-coupled receptor used in the embodiment 3 means any agonist that interacts with the G protein-coupled receptor or any antagonist of the receptor.

<Optical imaging>
The optical imaging in the aspect 3 means an imaging method for monitoring, recording and analyzing the presence, absence or intensity of a detectable signal emitted by the reporter gene in a cell into which the reporter gene has been introduced. In order to achieve optical imaging, the intensity of the signal emitted by the reporter gene must be high enough so that the signal can be analyzed from outside the cell. Since optical imaging is readily applicable to automation, it can be used to monitor multiple gene expressions simultaneously.

B. Optical imaging method for gene expression <Production of gene expression vector>
When using animal cells, the expression vector preferably contains at least a promoter, a start codon, DNA encoding the protein of interest, and a stop codon. Also includes DNA encoding signal peptide, enhancer sequence, 5 'and 3' untranslated regions of gene encoding the protein, splicing junction, poly A addition signal, selectable marker region or replicable unit, etc. You may go out.

<Gene transfer method into cells>
Examples of methods for introducing genes into cells include calcium chloride method or calcium chloride / rubidium chloride method, lipofection method, electroporation method and the like. The cells into which the gene has been introduced used in Aspect 3 include both transiently expressing cells and stable expressing cells.

<Optical imaging of gene expression by stimulation>
Aspect 3 can be implemented as follows, for example.

A reporter gene (preferably a firefly or a reporter gene) operably linked to a cell constant (for example, a promoter region of a gene whose expression is induced by stimulation with which the substance is brought into contact with the cell (preferably, the promoter region of the c-fos gene)) The luciferase derived from Renilla, etc. is introduced into the cells using the above-described gene introduction method, and constants of the obtained cells into which the genes have been introduced (for example, 1 to 1 × 10 ⁹ cells, preferably 1 × 10 ^3). ˜1 × 10 ⁶ ) are cultured in a desired nutrient medium (eg, D-MEM medium) using an apparatus capable of culturing the desired cell (eg, petri dish, multi-plate having many wells). The sample consisting of this constant number of cells is preliminarily kept at a temperature optimum for the cells (for example, 25 to 37 ° C., preferably 35 to 37 ° C.), and water is injected to prevent the sample from drying. A light emitting image is recorded with a digital camera through an objective lens in a sample observation part of the light emission microscope, which is placed in a culture device part of a wet light emission microscope. For example, a compound) is added at a desired concentration (eg, 1 pM to 1 M, preferably 100 nM to 1 mM), and a luminescence image is recorded at a desired time interval (eg, 5 minutes to 5 hours, preferably 10 minutes to 1 hour). The brightness value in a desired area in the image is acquired from the recorded image using commercially available image analysis software (for example, MetaMorph; manufactured by Universal Imaging Co., Ltd.). Record and superimpose the luminescent image and the bright field image using the image analysis software described above to identify the luminescent cells.

[Example 1]
<Construction of c-fos promoter-luciferase reporter gene>
A c-fos promoter-luciferase reporter gene was constructed as follows in accordance with the method described in Japanese Patent Application No. 2005-352683 (reference number 05P03010). Using human genomic DNA as a template, PCR was performed using primer fos pro Fw and primer fos pro Rv to amplify the promoter region of the c-fos gene (-363 to +1,067 with respect to the transcription start site). The amplified DNA fragment was digested with restriction enzymes XhoI and HindIII. The expression vector pGL3-Basic vector (Promega) was digested with restriction enzymes XhoI and HindIII, and the above DNA fragment was cloned into the pGL3-Basic vector. (Hereinafter, it may be described as a reporter gene expression vector (pGL3-fos).)
[Example 2]
<Change in expression level of reporter gene when cells are stimulated with ATP as a ligand>
An example of measuring the change in the expression level of a reporter gene when ATP is used as a ligand for cell stimulation is shown below.

HeLa cells were spread on a glass bottom dish (diameter 35 mm; manufactured by IWAKI) at 1 × 10 ⁵ cells / dish. D-MEM containing 10% fetal bovine serum was used as the growth medium. After culturing the cells at 37 ° C. for 1 day, a reporter gene expression vector was introduced into the cells using Lipofectamine 2000 reagent (Invitrogen) according to the manufacturer's instructions. After about 2 hours, the gene transfer solution was discarded, and a growth medium was added, followed by culturing at 37 ° C. for 16 hours or more. The growth medium was replaced with a serum-free medium (D-MEM not containing fetal calf serum) and cultured at 37 ° C. for 16 hours or more. The incubator in which the cells were cultured was placed in a culture apparatus part of a luminescence microscope that had been set to 35 to 37 ° C. in advance, and the sample observation part was operated to focus the cells. Luciferin was added to a final concentration of 1 mM and allowed to stand for 1 hour or longer, and then ATP was added to a final concentration of 100 μM. Using a digital camera (DP30; Olympus) operation software (DP controller; Olympus), bright-field images of the cells were captured and recorded. The luminescence image was obtained by adding 5 times of exposure to a digital camera for 1 minute. Luminescent images obtained every 10 minutes were captured by a computer and recorded. The luminance value of the luminescent image was obtained using image analysis software (Metamorph; manufactured by Universal Imaging), and the luminescent image and the bright field image were superimposed to identify the luminescent cells.

  FIG. 10-3 shows a superimposed image when identification is performed in the second embodiment. Here, FIG. 10-1 shows a bright-field image of a cell, FIG. 10-2 shows a luminescence image acquired in the same field, and FIG. 10-3 shows an image obtained by superimposing the bright-field image and the luminescence image on image analysis software. It was. From this superimposed image, the position and form of the luminescent cells could be confirmed.

  As shown in FIG. 11, it was observed that the activity of luciferase, which is a reporter gene product, was transiently increased in response to a stimulus in contact with ATP in a single cell. However, it was found that the time required for the luciferase activity to reach its peak after stimulation and the amount of activity at the peak varied between cells.

[Example 3]
<Change in expression level of reporter gene when cells are stimulated with serum>
An example of measurement of change in the expression level of a reporter gene when fetal bovine serum is used as a ligand for cell stimulation is shown below. Other conditions were the same as in Example 2 except that the ligand used was different. Fetal calf serum stimulation was performed at a final concentration of 10%.

  FIG. 12C shows a superimposed image when identification is performed in the third embodiment of the third aspect. Here, a bright field image of a cell is shown in FIG. 12-1, a luminescence image acquired in the same field is shown in FIG. 12-2, and an image obtained by superimposing the bright field image and the luminescence image on image analysis software is shown in FIG. It was. The position and form of the luminescent cells were confirmed.

  As shown in FIG. 13, it was observed in single cells that the activity of luciferase, a reporter gene product, increased transiently in response to stimulation with fetal calf serum. However, it was found that the time required for the luciferase activity to reach a peak after stimulation and the amount of activity at the peak varied between cells, similar to the ATP stimulation in Example 2.

  According to the description described in Examples 1 to 3 above, it can be understood that the aspect 3 is an invention expressed in the following additional items.

[Additional Item 1]
A method for analyzing changes in gene expression level in response to a stimulus in a single cell,
Introducing into the cell a reporter gene operably linked to the promoter region of the gene whose expression is induced by the stimulus of contacting a substance with the cell;
Maintaining the cells in a state where they can continue to survive;
A step of stimulating cells by contacting the substance with the substance,
Optical imaging of a detectable signal produced by the reporter gene expressed in each stimulated cell;
Quantitatively determining the amount of signal generated by the reporter gene at a high S / N ratio;
A method for analyzing a change in gene expression level in a single cell, comprising a step of identifying a cell in which a signal generated by the reporter gene is detected.
[Additional Item 2]
Item 2. The method according to Item 1, wherein the promoter region is a promoter region of an early gene.
[Additional Item 3]
Item 3. The method according to Item 2, wherein the earliest gene is a human-derived c-fos gene.
[Additional Item 4]
Item 2. The method according to Item 1, wherein the reporter gene is a photoprotein.
[Additional Item 5]
Item 5. The method according to Item 4, wherein the photoprotein is firefly-derived luciferase.
[Additional Item 6]
Item 5. The method according to Item 4, wherein the photoprotein is a Renilla luciferase.
[Additional Item 7]
Item 2. The method according to Item 1, wherein the reporter gene operably linked to the promoter region is introduced into a cell.
[Additional Item 8]
Item 2. The method according to Item 1, wherein the substance brought into contact with the cell is a ligand of a G protein-coupled receptor.
[Additional Item 9]
Item 9. The method according to Item 8, wherein the ligand of the G protein-coupled receptor is a nucleic acid.
[Additional Item 10]
Item 10. The method according to Item 9, wherein the nucleic acid is adenosine triphosphate.
[Additional Item 11]
Item 2. The method according to Item 1, wherein the substance to be contacted with cells is serum.
[Additional Item 12]
Item 2. The method according to Item 1, wherein a signal generated by the reporter gene is optically imaged.
[Additional Item 13]
Item 13. The method according to item 12, wherein imaging is performed with a light emission microscope in the optical imaging.
[Additional Item 14]
Item 2. The method according to Item 1, wherein the intensity of the signal generated by the reporter gene is converted into a luminance value.
[Appendix 15]
Item 2. The method according to item 1, wherein a bright-field image is captured with a light emission microscope in the cell.
[Additional Item 16]
Item 2. The method according to Item 1, wherein a cell producing a signal is identified by superimposing an optical imaging image of a signal generated by the reporter gene and a bright field image of the cell.

[Aspect 4] Method for analyzing gene expression in specific cells [Technical Field]
This aspect 4 relates to a method of analyzing gene expression in specific cells in a biological sample containing a plurality of cells in the present invention.

[Background technology]
In the conventional promoter assay method, it was impossible to see that the change in gene expression level with respect to external stimuli was different depending on individual cells. However, the response to stimuli is more complex in heterogeneous cell populations, as it is known that the response of individual cells to stimuli is different even when using cell lines with relatively uniform properties. Is expected. Furthermore, conventionally, since an experimental system in which uniform cells are gathered was used, it was not necessary to identify individual cells, and the average value of all cells was observed. However, in experimental systems using heterogeneous cell populations such as primary culture and slice culture, signals from the target cells overlap with the signals of surrounding cells using the conventional promoter assay method to accurately identify the cells. I can't. In addition, early genes (such as c-fos) whose expression is induced by stimulation from outside the cell are known to be expressed in many cell types. It is possible to observe the reactivity of cells to the real time. However, in the presence of multiple types of cells, it is difficult to identify cells that have been expressed. Therefore, it is necessary to construct an experimental system in which changes in the expression level of the initial gene can be seen in real time only in specific cells.

[Problems arising from the above background]
Conventional methods do not consider observing gene expression only in specific cells in a group of cells consisting of multiple cell types, and optical imaging of detectable signals generated by luciferase genes only from specific cells I couldn't cope with that. The present invention pays attention to this point, and it is an object to provide a method for analyzing gene expression in specific cells, in which a signal generated by a luciferase gene is emitted only in a specific cell, and the signal is optically imaged with a light emission microscope. It becomes.

[Means and effects for solving the above problems]
One of the split luciferases fused to the MyoD gene or Id gene is expressed only in specific cells using the cell-specific promoter region, and the other uses the promoter region of the gene (such as the first-stage gene) whose expression level is to be analyzed. To express in cells. By observing this cell sample with a light emission microscope, it is possible to optically image specific cells in which both fusion genes are expressed and the enzyme activity of luciferase is observed.

  In carrying out the present invention, in the patent (US Pat. No. 5,670,356) and non-patent paper (PNAS (2002) vol.99 No.24 pp3105-3110), the protein protein in the cell using the mammalian two-hybrid system. Reference can be made to the detection method and the promoter assay method. This is a fusion protein expression vector of genes known to cause interactions (MyoD gene and Id gene) and yeast GAL4 DNA binding domain or herpes simplex virus VP16 activation domain, and five GAL4 binding regions The contained luciferase expression vector is introduced into the cells. Protein interaction detection and promoter assay are performed using the principle that the expression level of luciferase increases when the target genes (MyoD gene and Id gene) interact with each other.

  In addition, a non-patent paper (PNAS (2002) vol.99 No.24 pp15608-15613) can also be referred to a method for detecting an interaction between proteins in a cell and a promoter assay method using split luciferase. This divides the luciferase gene into an N-terminal side and a C-terminal side, and creates a fusion protein with genes (MyoD gene and Id gene) that are known to cause interaction and expresses them in cells. Luciferase has no enzymatic activity in the N-terminal or C-terminal state only, so no luminescence is detected. However, if there is a bond between the fused genes (MyoD gene and Id gene), the N-terminal side and C-terminal side of luciferase Are used to detect interactions between proteins and to perform promoter assays using the principle that they also bind and have enzyme activity.

[A. Definition of terms in aspect 4]
<Promoter region of gene expressed in specific cells>
Examples of the promoter region of a gene expressed in a specific cell in Embodiment 4 include a neuron-specific gene or a glial cell-specific gene promoter.

  Examples of the promoter for a neuron-specific gene used in Aspect 4 include the promoter region of an NSE (neuron-specific enolase) gene. Examples of the glial cell-specific gene promoter used in aspect 4 include the promoter region of a GFAP (glial fibrillary acidic protein) gene. The promoter used in aspect 4 includes any of the above promoters. Also includes counterparts of animal species. Here, the promoter region means an arbitrary region containing the minimum base sequence necessary for having promoter activity. For example, a part or all of the region of 500 to 4000 bases upstream from the transcription site of the gene can be used.

<Promoter region of a gene whose expression is induced by stimulation>
Examples of the gene promoter region whose expression is induced by stimulation in aspect 4 include a promoter of an early gene. Examples of the promoter of the early gene used in aspect 4 include the c-fos promoter region. In addition, the promoter used in aspect 4 includes the counterpart of any animal species of the promoter. Here, the promoter region means an arbitrary region containing the minimum base sequence necessary for having promoter activity. For example, a part or all of the region of 500 to 2000 bases upstream from the transcription site of the gene can be used.

<Reporter gene>
The reporter gene in embodiment 4 means a gene encoding a reporter protein that emits detectable fluorescence. For example, luciferase derived from firefly or Renilla can be mentioned. Furthermore, the gene which codes (beta) galactosidase and the gene which codes alkaline phosphatase can be mentioned, for example.

<Substance>
The substance used in the aspect 4 means a natural substance existing in nature or any substance prepared artificially. Specifically, for example, any compound synthesized chemically can be mentioned. The type and molecular weight of the compound are not particularly limited. When the substance is a protein or peptide, those isolated from living tissues and cells and those prepared by genetic recombination or chemical synthesis are also included. Furthermore, those chemical modifications are also included.

<Optical imaging>
The optical imaging in the aspect 4 means an imaging method for monitoring, recording and analyzing the presence, absence or intensity of a detectable signal emitted by the reporter gene in a cell into which the reporter gene has been introduced. In order to achieve optical imaging, the intensity of the signal emitted by the reporter gene must be high enough so that the signal can be analyzed from outside the cell. Since optical imaging is readily applicable to automation, it can be used to monitor multiple gene expressions simultaneously. Note that the technology for processing image information two-dimensionally or three-dimensionally at an arbitrary position of an imaged sample image can also be referred to Japanese Patent Application Nos. 2004-172156 and 2004-178254 by the present applicant. Good. The difference between time-series image acquisition in fluorescence observation and light emission observation is that there is no influence of extra light because light emission observation does not require optical scanning (laser scan) with excitation light. According to the imaging technique performed in Aspect 4, a luminescent image can be captured in real time.

B. Optical imaging method for gene expression <Production of gene expression vector>
When using animal cells, the expression vector preferably contains at least a promoter, a start codon, DNA encoding the protein of interest, and a stop codon. Also includes DNA encoding signal peptide, enhancer sequence, 5 'and 3' untranslated regions of gene encoding the protein, splicing junction, poly A addition signal, selectable marker region or replicable unit, etc. You may go out.

<Gene transfer method into cells>
Examples of methods for introducing genes into cells include calcium chloride method or calcium chloride / rubidium chloride method, lipofection method, electroporation method and the like. The gene-introduced cell used in aspect 4 includes both transiently expressed cells and stablely expressed cells.

<Optical imaging of gene expression by stimulation>
For example, Embodiment 4 can be implemented as follows.

  N-terminal luciferase (NLuc) gene (amino acid numbers 1-416) and mouse Id gene (amino acid numbers 29-148) fusion gene (NLuc-Id gene), and C-terminal luciferase (CLuc) gene (amino acid number) 398-550) and a mouse MyoD gene (amino acid numbers 1-318) are prepared (MyoD-CLuc gene). As shown in FIG. 14, when the Id gene site in the NLuc-Id gene expressed in the cell and the MyoD gene site in the MyoD-CLuc gene interact and bind to each other, the NLuc gene and the CLuc gene bind and detect. Emits possible signals.

  In addition, cell-specific expression is performed together with the NLuc-Id gene operably linked to the promoter region of the early gene (for example, the promoter region of the c-fos gene) whose expression is induced by stimulation with the substance contacting the cell. If you want to observe neurons, use the promoter region of the NSE (neuron-specific enolase) gene that is expressed specifically in nerve cells. If you want to observe glial cells, A cell group consisting of a plurality of cell types obtained by mixing and culturing MyoD-CLuc gene operably linked to a GFAP (glial fibrillary acidic protein) gene expressed using a conventional gene transfer method (for example, Introduced into a mixed culture of nerve cells and glial cells). Alternatively, the promoter region of a gene that expresses cell-specifically together with the MyoD-CLuc gene operably linked to the promoter region of the earliest gene (for example, the promoter region of the c-fos gene) In some cases, the NSE (neuron-specific enolase) gene promoter region that is expressed specifically in nerve cells is used. To observe glial cells, the GFAP (glial fibrillary acidic protein) gene promoter region that is specifically expressed in glial cells The NLuc-Id gene operably linked to the cell group (for example, a mixed culture of nerve cells and glial cells) is introduced into the cell group consisting of a plurality of cell types mixed and cultured using a conventional gene transfer method.

The number of cells into which the gene has been introduced (for example, 1 to 1 × 10 ⁹ cells, preferably 1 × 10 ³ to 1 × 10 ⁶ cells) can be used for a desired cell culture (for example, petri dish, multiple wells) In a desired nutrient medium (for example, D-MEM medium) using a multiplate or the like. A sample of this constant number of cells is preliminarily kept at a temperature optimum for the cells (for example, 25 to 37 ° C., preferably 35 to 37 ° C.) and water is injected to prevent the sample from being dried. A luminescent image is recorded by a digital camera through an objective lens in a sample observation section of the luminescence microscope that is installed in the culture apparatus. A substance (for example, a compound) for stimulating by contacting cells is added to the sample at a desired concentration (for example, 1 pM to 1 M, preferably 100 nM to 1 mM), and a desired time interval (for example, 5 minutes) is added. -5 hours, preferably 10 minutes to 1 hour). As shown in FIG. 15, in a specific cell B in which the NLuc-Id gene (or MyoD-CLuc gene) is expressed, the expression of the MyoD-CLuc gene (or NLuc-Id gene) by stimulation with contacting the substance with the cell When is induced, the enzyme activity of luciferase is enhanced and a detectable signal is emitted. On the other hand, no detectable signal is emitted in cell A in which the expression of only the MyoD-CLuc gene (or NLuc-Id gene) has been induced by the stimulation of contacting the substance with the cell. The recorded image uses a commercially available image analysis software (for example, MetaMorph; manufactured by Universal Imaging Co., Ltd.) to obtain a luminance value in a desired area in the image. Furthermore, a bright field image is recorded in the same field as the luminescent image, and the luminescent image and the bright field image are superimposed using the image analysis software described above to identify the luminescent cells.

According to the description of the above-described embodiment regarding this application example, it can be understood that the aspect 4 is an invention expressed in the following supplementary items.
[Additional Item 17]
A method of analyzing gene expression in specific cells,
Introducing into the cell an incomplete reporter gene operably linked to a promoter region of a gene expressed in a specific cell;
Introducing into the cell an incomplete reporter gene operably linked to the promoter region of the gene whose expression is induced by the stimulus of contacting a substance with the cell;
Maintaining the cells in a state where they can continue to survive;
A step of stimulating cells by contacting the substance with the substance,
Optically imaging a detectable signal generated by binding of the incomplete reporter genes expressed in each stimulated cell;
Quantitatively determining the amount of signal generated by the reporter gene at a high S / N ratio;
A method for analyzing gene expression in a specific cell, comprising a step of identifying a cell in which a signal generated by the reporter gene is detected.
[Additional Item 18]
Item 18. The method according to Item 17, wherein the promoter region of a gene expressed in the specific cell is a promoter region of a gene expressed specifically in a nerve cell.
[Appendix 19]
Item 19. The method according to item 18, wherein the gene that is specifically expressed in nerve cells is an NSE (neuron-specific enolase) gene.
[Appendix 20]
Item 18. The method according to Item 17, wherein the promoter region of a gene expressed in the specific cell is a promoter region of a gene expressed specifically in glial cells.
[Appendix 21]
Item 18. The method according to Item 17, wherein the glial cell-specific expression gene is a GFAP (glial fibrillary acidic protein) gene.
[Appendix 22]
Item 18. The method according to Item 17, wherein the promoter region whose expression is induced by the stimulation is a promoter region of an early gene.
[Additional Item 23]
Item 22. The method according to Item 22, wherein the earliest gene is a human-derived c-fos gene.
[Appendix 24]
Item 18. The method according to Item 17, wherein the incomplete reporter gene comprises a part of the gene sequence of the photoprotein.
[Appendix 25]
Item 25. The method according to Item 24, wherein the photoprotein is firefly-derived luciferase.
[Appendix 26]
Item 25. The method according to Item 24, wherein the photoprotein is a Renilla luciferase.
[Appendix 27]
Item 18. The method according to Item 17, wherein the incomplete reporter gene operably linked to the promoter region is introduced into a cell.
[Appendix 28]
Item 18. The method according to Item 17, wherein the substance that contacts the cell is a ligand of a G protein-coupled receptor.
[Appendix 29]
Item 18. The method according to Item 17, wherein the substance to be brought into contact with cells is serum.
[Appendix 30]
Item 18. The method according to Item 17, wherein a signal generated by combining the incomplete reporter genes is optically imaged.
[Appendix 31]
Item 31. The method according to Item 30, wherein imaging is performed with a light emission microscope in the optical imaging.
[Appendix 32]
Item 18. The method according to Item 17, wherein the intensity of the signal generated by the reporter gene is converted into a luminance value.
[Additional Item 33]
Item 18. The method according to Item 17, wherein a bright-field image is captured with a light emission microscope in the cell.
[Additional Item 34]
Item 18. The method according to Item 17, wherein the cell producing the signal is identified by superimposing an optical imaging image of the signal generated by the reporter gene and a bright field image of the cell.

[Aspect 5] Method for detecting luciferase activity expressed on the surface of a cell [Technical field of aspect 5]
This aspect 5 relates to a method for analyzing the secretion of a protein outside the cell or the expression on the cell surface in the present invention. More specifically, a signal peptide predicted from a sequence rich in hydrophobic amino acids or a gene encoding a transmembrane domain encoding a fusion protein using luciferase is expressed on the surface of a cell by an analysis method using a light emission microscope or the like. And a method for detecting luciferase activity.

[Background technology]
In multicellular animals including humans, it is necessary to establish intercellular communication and extracellular environment for the construction and maintenance of individuals. For this purpose, cells secrete a huge variety of proteins out of the cell through an organelle called the endoplasmic reticulum-Golgi apparatus. For example, secretory water-soluble proteins such as hormones are secreted outside the cell through the cell membrane, and the membrane protein is incorporated into an appropriate membrane such as a cell membrane. Secreted proteins have a sequence called “secreted signal peptide” at the N-terminus. As soon as the signal peptide is first translated in the cytoplasmic ribosome, it is inserted into a channel (hole) in the membrane of the endoplasmic reticulum by a group of molecules that recognize it, and then passes through the protein body part that is synthesized, It is disconnected and destroyed. The signal peptide is a sequence rich in hydrophobic amino acids, and there is a program that can predict this with high probability.

  For example, there is a technique for discriminating the presence and the region of a signal peptide with respect to an arbitrary amino acid sequence. The outline of the discrimination algorithm is that the average hydrophobicity value exceeds a certain threshold in the input amino acid sequence and includes negatively charged residues using the hydrophobicity index and negative charge index unique to each amino acid. A region that is not present is searched, and the region closest to the N-terminal is used as a candidate region for the signal peptide. Next, the discrimination region of the signal peptide is set using the position having the maximum average hydrophobicity value in the candidate region, and the position of the positively charged residue closest to the N-terminal side (N-terminal in the absence). This discrimination area often does not match the candidate area. Two indices SS-index and SP-index for discriminating the newly invented signal peptide are allocated to amino acids constituting the discrimination region. Whether or not the candidate region is a signal peptide is determined from a discriminant that takes these values and position information of the candidate region into consideration. The SOSUIsignal prediction program focuses on the physicochemical characteristics found in the amino acid sequences of the three domain structures. Its prediction system detects highly hydrophobic segments, predicts signal sequences including signal anchors, signals It consists of three modules: discrimination between anchor and signal peptide. By detecting these signal peptides, (1) analysis of proteome information, estimation of new gene / protein functions revealed by the progress of genome planning, and (2) a large amount of biological information using bioinformatics computers , (3) Genome drug discovery, and use of signal peptides to improve production technology for protein preparations such as lysozyme and hormones.

  In addition, cytokines play an important role in biological defenses such as immune responses, inflammation, and hematopoietic reactions, and search for new cytokines or cytokine-like bioactive protein genes to elucidate their structures and functions. This is also very important for elucidating the cause of the disease and applying it to treatment. Furthermore, if the target protein can be secreted into the culture supernatant, purification can be facilitated and the production of recombinant protein in the host cell can be greatly increased. The cost reduction of the recombinant protein preparation will be realized.

[Problems arising from the above background]
The prediction and function of these signal peptides are finally verified using cells. As a conventional method, the gene trap method introduces a reporter gene lacking a promoter into a target cell and suppresses endogenous gene expression by the reporter activity when inserted downstream of the promoter activated on the chromosome. To identify. For example, the reporter is a fusion gene of β-balactosidase (β-gal) and (hygromycin (Hyg.) Phosphotransferase. If this gene is translated and the reporter protein is synthesized and stays in the cell, β-gal activity is expressed and X A region that is stained blue in the cytoplasm appears in the presence of -gal, but its activity is lost when entering the ER side, a hydrophobic transmembrane region is incorporated upstream of the reporter, and a signal sequence is defined using β-gal activity as an indicator. The gene to be encoded can be selected and the secretory type and membrane surface protein specific to chondrocytes can be identified by the gene trap method targeting these signal sequences (Proc. Natl. Acad. Sci. USA Vol. 92, pp.6592-6596) However, in the presence of a signal peptide, β-balactosidase activity is lost, so there are problems that false positives increase and detection sensitivity is not good.

  In addition, as a method using bioluminescence such as fluorescent protein and luciferase, the culture supernatant after gene transfection is collected, and fluorescence of the fluorescent protein in the culture supernatant and luminescence due to luciferase activity are detected. We are identifying unknown secreted proteins. However, since the culture supernatant is collected, there is a problem that the sensitivity becomes low, the cell is secreted, and the intensity of expression cannot be measured at the cell level.

  Here, in the prior art using a fluorescent protein, the phototoxicity to cells by irradiation with excitation light is not considered, and it is not possible to cope with the point of maintaining cells in a state where they can continue to survive. Therefore, paying attention to this point, it is an object to provide a method for analyzing gene expression in a single cell using bioluminescence, in which there is no need to irradiate excitation light, and thus there is little damage to cells. In addition, the conventional technology using fluorescent proteins does not consider the background of excitation light itself or autofluorescence of intracellular substances, and quantitatively determines the amount of signal generated by the reporter gene with a high S / N ratio. I cannot cope with the point of doing. Therefore, it is also an object to provide a method for analyzing gene expression in a single cell using bioluminescence, which uses a photoprotein that does not require excitation light and has a relatively low background.

  In addition, in the observation by immunostaining method, the cells cannot be fixed and must be stained in the state of living cells, and in the case of adhesive cells, it takes time and labor because the cells are peeled off from the plate or accompanied by a washing operation. There is a problem. Furthermore, when observing the expression on the cell surface, a method of observing with a protein in which a transmembrane domain is fused to GFP or the like has been attempted, but it is difficult to determine whether it is expressed outside the cell. There is also a problem that there are many false positives. Specifically, a fusion protein containing a transmembrane domain from 187 amino acids to the C terminus of CD40 was transfected into the C terminus of GFP. Hela cells expressing this fusion protein were observed by immunostaining with anti-GFP antibody (sigma) and anti-mouse-IgG-Cy3 (sigma) as a secondary antibody in an environment of 4 ° C. Then, cells expressed on the cell surface were observed despite the absence of signal peptide by immunostaining (FIG. 16).

  Furthermore, in the prior art using a photoprotein, it is not considered to observe a signal generated by a reporter gene in a single cell, and it is possible to cope with optical imaging of a detectable signal generated by a reporter gene. I can't. Therefore, focusing on this point, it is possible to provide a method for analyzing gene expression in a single cell using bioluminescence, in which imaging is performed with a luminescence microscope so that cells emitting a signal generated by a reporter gene can be identified. It becomes a problem.

[Means and effects for solving the above problems]
1. In the prior art, as described above, any of the conventional analysis methods related to signal peptide detection can observe the presence or absence of a signal peptide for each cell in the method using β-balactosidase activity. However, it is not directly known whether it is released to the outside of the cell, and it has the problem of low sensitivity because it uses β-balactosidase activity as an index. In addition, in the method of measuring the culture supernatant in which GFP or luciferin is secreted from the cells, the culture supernatant must be concentrated in order to increase the sensitivity because it is not possible to directly observe the cells and only the information of the whole culture medium to be measured is known. There is a problem that must be.

  Aspect 5 can be detected with high sensitivity by retaining the reporter on the extracellular membrane for measurement such as extracellular secretion. In other words, a gene that observes bioluminescence, such as a luciferase that has almost no substrate in the cell, is connected to the C terminus of the signal peptide to be analyzed, and a reporter gene that is connected to a known protein that remains on the cell surface at the C terminus. By constructing and observing with a light emission microscope, it is possible to obtain individual information with high sensitivity to the action of the signal peptide.

  The biosynthesis of GPI anchors can be summarized as follows. The first step is the transfer of / N / -acetylglucosamine from UDP- / N / -acetylglucosamine to phosphatidylinositol. The product / N / -acetylglucosamine-phosphatidylinositol is then deacetylated to produce glucosamine-phosphatidylinositol. This reaction produces unacetylated glucosamine that is very specific for GPI. Studies of GPI-anchored biosynthetic mutants of mammalian cells have shown that glucosamine-phosphatidylinositol biosynthesis involves at least three genes. Addition of mannose, which forms three mannose chains conserved as a core structure, occurs by the formation of a hydrophobic donor by the transfer of GDP-mannose to activated mannose to dolichol mannose phosphate. Next, ethanolamine phosphate is added to the third mannose (counted from the reducing end of the core glycan) by the hydrophobic donor phosphatidylethanolamine, thus completing the conservative core glycan biosynthesis.

  The transamidase reaction that occurs on the luminal side of the endoplasmic reticulum is initiated as soon as the protein is inserted into the appropriate location in the endoplasmic reticulum membrane. It is necessary for the subsequent GPI anchoring that the precursor protein chain penetrates the endoplasmic reticulum membrane. This reaction itself is a transamination type reaction in which the C-terminal of the protein is cleaved and replaced by a GPI anchor through the amino group of ethanolamine phosphate. GPI-anchored proteins interact with GPI-transamidase on the lumen side of the endoplasmic reticulum. This enzyme recognizes a hydrophobic amino acid chain extending to the C-terminal side. This amino acid chain is assumed to temporarily function as a transmembrane region, and a previously generated GPI anchor is attached to a new C-terminal amino acid generated by transamidase cleavage of this amino acid chain.

  The most obvious function of a protein-bound GPI is to localize a specific protein outside the cell membrane, usually toward the extracellular environment, with a stable structure to the membrane. This may be a general reason why this protein is GPI-anchored, unlike proteins that are tethered through the transmembrane region. One of the main advantages of being GPI-anchored is the physical sequestration of the surface protein that functions outside the cell from the cytoplasm, thereby protecting the inside of the cell. GPI makes it possible to assemble surface molecules at an extremely high density that is unthinkable for transmembrane proteins, thereby allowing complete isolation from the hostile environment. Specificity can be increased by using a GPI anchor type for such a known protein remaining on the cell surface.

  In addition, a reporter gene that connects to the C terminus of a known signal peptide, and a protein that remains on the cell surface to be analyzed is constructed at the C terminus, and the action of the transmembrane domain and GPI anchor type is observed by observing with a light emission microscope. It is possible to obtain individual information with high sensitivity. Since it is possible to observe individual cell information with high sensitivity, it is possible to simultaneously analyze whether different signal peptides or transmembrane domains in the same container, and it is possible to reduce the required amount of sample and the number of reaction containers. Can be simplified.

[Aspect]
The N-terminus of the Renilla Luciferase gene connected to the rat neurotrophin-3 (NT-3) signal peptide part-MSILFYVIFLAYLRGI- (sig pep-Luci-GPI) and the one not connected (Luci-GPI) to the C-terminus In addition, a gene expressing a fusion protein was constructed in which a portion of human Thy-1 from the N-terminus to 117 (Ser) -161 (Leu) was connected.

First, the day before transfection, 2 ml (D-MEM medium containing 10% FBS) was seeded at a concentration of 5 × 10 5 cells / ml in a 35 mm culture dish and cultured at 37 ° C. in a 5% CO ₂ environment. On the next day, a plasmid in which the sig pep-Luci-GPI or Luci-GPI gene was inserted into the pDNA3.1 expression vector was transfected into Hela cells using Lipofectamine 2000 (Invitrogen). 24 to 48 hours after transfection, the cells were cultured in D-MEM medium containing 10% FBS at 37 ° C. in a 5% CO ₂ environment.

  For the measurement, the cells were washed with HANKS, 5 μl of Renilla Luciferase Assay Substrate (100 ×) (Promega) was added to 0.5 ml of D-MEM medium, and observed using a luminescence microscope (see FIGS. 17 and 18). The measurement conditions were that after taking a bright field image, a luminescence image was taken with an exposure time of 5 minutes and an objective magnification of 20 times. As shown in FIG. 17A, sig pep-Luci-GPI showed significant luminescence in the image observed on the luminescence (right side of the figure) corresponding to the image by bright field observation (left side of the figure). On the other hand, as shown in FIG. 17-2, in the luminescence observed image (right side of the figure) corresponding to the bright field observation image (left side of the figure), Luci-GPI without the signal peptide emits almost luminescence. I was not able to admit.

  The expression of the gene was measured with a Luminescencer-JNRII (ATTO) luminometer after lysing the cells according to the pro and call of Renilla Luciferase Assay System (Promega) (see FIG. 18). The measurement time was 10 seconds, and luminescence was measured for both sig pep-Luci-GPI and Luci-GPI, and it was confirmed that there was no problem in the expression of luciferase.

[A. Definition of words]
<Reporter gene>
The reporter gene in aspect 5 means a gene encoding a reporter protein that emits detectable fluorescence. For example, luciferase derived from firefly or Renilla can be mentioned. Furthermore, the gene which codes (beta) galactosidase and the gene which codes alkaline phosphatase can be mentioned, for example.

<Promoter region of a gene whose expression is induced by stimulation>
Examples of the promoter region in Aspect 5 include a promoter of an early gene.

  Examples of the promoter of the earliest gene used in Aspect 5 include the c-fos promoter region. In addition, the promoter used in aspect 5 includes the counterpart of any animal species of the promoter. Here, the promoter region means an arbitrary region containing the minimum base sequence necessary for having promoter activity. For example, a part or all of the region of 500 to 2000 bases upstream from the transcription site of the gene can be used.

<Substance>
The “substance” used in the invention described below means a natural substance existing in nature or any substance prepared artificially. Specifically, for example, any compound synthesized chemically can be mentioned. The type and molecular weight of the compound are not particularly limited. When the substance is a protein or peptide, those isolated from living tissues and cells and those prepared by genetic recombination or chemical synthesis are also included. Furthermore, those chemical modifications are also included.

<Production of gene expression vector>
When using animal cells, the expression vector preferably contains at least a promoter, a start codon, DNA encoding the protein of interest, and a stop codon. Also includes DNA encoding signal peptide, enhancer sequence, 5 'and 3' untranslated regions of gene encoding the protein, splicing junction, poly A addition signal, selectable marker region or replicable unit, etc. You may go out.

<Gene transfer method into cells>
Examples of methods for introducing genes into cells include calcium chloride method or calcium chloride / rubidium chloride method, lipofection method, electroporation method and the like. The gene-introduced cell used in aspect 5 includes both transiently expressed cells and stablely expressed cells.

<Optical imaging of gene expression by stimulation>
For example, the embodiment 5 can be implemented as follows.

A reporter gene (preferably a firefly or a firefly The luciferase derived from Renilla, etc. is introduced into the cells using the above-described gene introduction method, and constants of the obtained cells into which the genes have been introduced (for example, 1 to 1 × 10 ⁹ cells, preferably 1 × 10 ^3). ˜1 × 10 ⁶ ) are cultured in a desired nutrient medium (eg, D-MEM medium) using an apparatus capable of culturing the desired cell (eg, petri dish, multi-plate having many wells). The sample consisting of this constant number of cells is preliminarily kept at a temperature optimum for the cells (for example, 25 to 37 ° C., preferably 35 to 37 ° C.), and water is injected to prevent the sample from drying. A light emitting image is recorded with a digital camera through an objective lens in a sample observation part of the light emission microscope, which is placed in a culture device part of a wet light emission microscope. For example, a compound) is added at a desired concentration (eg, 1 pM to 1 M, preferably 100 nM to 1 mM), and a luminescence image is recorded at a desired time interval (eg, 5 minutes to 5 hours, preferably 10 minutes to 1 hour). The brightness value in a desired area in the image is acquired from the recorded image using commercially available image analysis software (for example, MetaMorph; manufactured by Universal Imaging Co., Ltd.). Record and superimpose the luminescent image and the bright field image using the image analysis software described above to identify the luminescent cells.

<Optical imaging>
The optical imaging in the aspect 5 means an imaging method for monitoring, recording and analyzing the presence, absence or intensity of a detectable signal emitted by the reporter gene in a cell into which the reporter gene has been introduced. In order to achieve optical imaging, the intensity of the signal emitted by the reporter gene must be high enough so that the signal can be analyzed from outside the cell. Since optical imaging is readily applicable to automation, it can be used to monitor multiple gene expressions simultaneously. Note that the technology for processing image information two-dimensionally or three-dimensionally at an arbitrary position of an imaged sample image can also be referred to Japanese Patent Application Nos. 2004-172156 and 2004-178254 by the present applicant. Good. The difference between time-series image acquisition in fluorescence observation and light emission observation is that there is no influence of extra light because light emission observation does not require optical scanning (laser scan) with excitation light. According to the imaging technique performed in Aspect 5, a luminescent image can be captured in real time. In the method of aspect 5, in particular, by using the light emission microscope as described above, it has become possible to realize the analysis based on the light emission image.

[Additional Item 1]
A method of detecting a signal peptide predicted from a sequence rich in hydrophobic amino acids, encoding a protein in the order of N-terminal to start codon, signal peptide to be analyzed, luminescence-related gene, known transmembrane domain, and stop codon. A process in which the DNA construct is introduced into a test cell, the encoded gene is transcribed and translated, and the protein molecule remains on the extracellular surface;
The process of detecting the luminescence activity of protein molecules expressed on the cell surface with a luminescence microscope, the process of analyzing the measurement results, the reaction solution and the reaction vessel that cause the above reaction, and observing and analyzing these cells A method comprising a method capable of determining a signal peptide from information.
[Additional Item 2]
A method for detecting a transmembrane domain predicted from a sequence rich in hydrophobic amino acids, encoding a protein in the order of N-terminal to start codon, known signal peptide, luminescence-related gene, transmembrane domain to be analyzed, and stop codon A process in which the DNA construct is introduced into a test cell, the encoded gene is transcribed and translated, and the protein molecule remains on the extracellular surface;
The process of detecting the luminescence activity of protein molecules expressed on the cell surface with a luminescence microscope, the process of analyzing the measurement results, the reaction solution and the reaction vessel that cause the above reaction, and observing and analyzing these cells A method comprising a method capable of determining a transmembrane domain from information.
[Additional Item 3]
A method of culturing cells simultaneously introduced with a DNA construct containing a DNA construct having a different base sequence of at least one signal peptide in the same container in the above method 1, and simultaneously analyzing other types of luminescent activity in the same container .
[Additional Item 4]
A detection method according to the above method 1, wherein a false positive due to a transmembrane domain can be opened by using a GPI anchor type for expression on a cell surface.
[Additional Item 5]
3. A detection method that makes it possible to discriminate between a reporter molecule expressed inside and outside a cell by using a substance in which the substrate does not easily enter the cell in the method 1 or 2.
[Additional Item 6]
The signal sequence in the said method or the screening method of a transmembrane domain.
[Additional Item 7]
A method for detecting the amount of secretion in the above method.

[Aspect 6] Induction of gene expression by tetracycline An example of a reporter assay over time in a single cell will be described.

  In this reporter assay, first, a vector “pcDNA6 / TR (manufactured by Invitrogen)” that constantly expresses tetracycline repressor (TetR) and an expression vector “pcDNA4 / TO (Invitrogen) having a tetracycline operator (TetO2)” are used. And a luciferase gene-linked plasmid are co-expressed in HeLa cells to prepare a specimen. In this state, since the TetR homodimer is bound to the TetO2 region, transcription of the luciferase gene is suppressed. Next, tetracycline is added to the culture solution to bind to the TetR homodimer, and by changing the three-dimensional structure of the TetR homodimer, the TetR homodimer is separated from TetO2 to induce transcription of the luciferase gene. The culture solution is a D-MEM medium containing 10 mM HEPES and contains 1 mM luciferin. Examples of the lens used as the objective lens and the imaging lens are commercially available objective lenses for microscopes having specifications of “Oil, 20 ×, NA 0.8” and “5 ×, NA 0.13”, respectively, and the magnification Mg The total magnification corresponding to is 4 times. The camera used as the camera C1 is a digital camera “DP30BW (manufactured by Olympus)” cooled at 5 ° C., and the CCD element as the CCD 3 is a 2/3 inch type, the number of pixels is 1360 × 1024, and the pixel size is 6 μm square. It is.

  A predetermined time after the addition of tetracycline, for example, 9 hours later, a self-luminous image of the specimen is exposed and imaged at appropriate intervals (for example, 1 minute), and 0 hours, 2 hours, 4 hours, and 6 hours after addition Observation images after 8 hours and 10 hours can be shown. A plurality of partial regions designated on the observation image (for example, for each of the four partial regions ROI-1, ROI-2, ROI-3, ROI-4, light is emitted based on the result of measuring the change in luminescence intensity of the luciferase gene over time. The position of the luciferase gene can be specified and traced over time (or time series), and the change in the luminescence phenomenon over time can be measured.

[Aspect 7] HSP70B gene promoter assay
HSP70B (Heat shock Protein70B) is a stress protein whose expression is induced by environmental factors such as heat, toxic substances, and oxygen deficiency. HSP not only contributes to the regulation of cell functions as a molecular chaperone, but also transmits intracellular information. It is also attracting attention as a substance. Therefore, the vector used in the reporter assay in this example was a vector in which the HSP70B promoter sequence was introduced into pGL3-Basic Vector (Promega). When stress such as heat shock is applied to the cells into which this vector has been introduced, the heat shock factor binds to the HSP70B promoter portion and transcription is initiated. HeLa cells cultured in a glass bottom dish were transfected with the vector by the lipofection method. After overnight culture, 1 mM Luciferin was added, and after heat stimulation (43 ° C., 60 minutes), time-lapse measurement was performed every 30 minutes in a luminescence microscope for 20 hours. As the medium, D-MEM5% FCS-10 mM HEPES was used. First, after obtaining the cell shape and position information by bright field observation, a luminescence image was obtained. The objective lens was a 20x lens, and the exposure time was 5 minutes.

  For the image obtained by superimposing the luminescent image and the bright field image, five regions (ROI) where one cell of interest enters were set, and the data was digitized and the average value was plotted. As a result, the promoter activity was highest at 10-15 hours after the heat shock, but there was a considerable difference in the rate of increase of the promoter activity and the time required for the activity increase among the 5 cells. In the observation with a conventional luminometer, since a certain number of cells are analyzed together, transfection efficiency, differences between cells, and the like are averaged. In reality, however, many untransfected cells were also present, and there were differences in expression depending on the cells. If data for each cell can be measured with a luminescence microscope, the analysis can be performed in consideration of the difference in transfection efficiency and the influence of the cell cycle.

[Aspect 8] Observation of intracellular localization Observation of intracellular localization in organelles was performed. HeLa cells were transfected with a pGL3 vector into which a signal sequence for localization to the nucleus, endoplasmic reticulum, and cell membrane was introduced. Was observed. A bright field image, a luminescent image, and an image obtained by superimposing the bright field and the luminescent image are shown. Localization to the target location was observed in each of the nucleus, endoplasmic reticulum, and cell membrane. If the intracellular localization can be detected, for example, it is possible to observe the transition from the cytoplasm to the nucleus when some stimulus is given to the cell. For example, detection of nuclear translocation or the like when a glucocorticoid receptor, which is an intranuclear receptor, is expressed and treated with a glucocorticoid hormone.

[Aspect 9] Analysis of interaction between different cells In the conventional luminescence detection, the amount of luminescence averaged by a luminometer was measured. It is also possible to analyze the interaction. An example is analysis of nerve cells and glial cells. The nervous system is mainly composed of two types of cells: neurons and glial cells, which play a central role in brain information processing through electrical activity and synaptic connections. Glial cells do not have such electrical activity and are thought to play a role in supporting nerve cell activity by surrounding nerve cells. Many of the roles of glial cells around nerve cells have not been clarified yet.

[Aspect 10] Luminescent reporter assay in tissue In addition to a luminescent reporter assay in cells, a target is a tissue sample. For example, by using a brain slice tissue, it is possible to capture different periods and phases for each cell in the suprachiasmatic nucleus, which is the place of a biological clock and brings a difference between day and night to the living thing.

  The present invention is not limited to the embodiments described above, and the following changes or improvements are possible. For example, although a photoprotein that functions as a gene is used as a luminescent substance, another bioluminescent substance having no gene function may be used. The present invention preferably includes a step of imaging while accumulating with a storage-type imaging device a photomarker substance that is minimal for the biological activity of a biological sample (for example, a cell or tissue). This imaging process obtains sample images with weak light that cannot be imaged with conventional scanning (scanning) high-intensity analysis such as fluorescence analysis. Emission analysis can be performed without giving Here, for a purpose different from light emission, a fluorescent substance or chemiluminescent substance having an extremely low concentration that is comparable to or less than half of the light emission may be used in combination. Further, the total amount per single sample may be distributed between the luminescent material and the fluorescent material at a desired ratio, and the total amount of luminescence may be adjusted to be the minimum necessary for storage-type imaging. Alternatively, cell recognition may be performed reliably by separately providing two or more colors of luminescent labels that do not overlap the nuclei and cytoplasm as much as possible. By recognizing the cytoplasm, various imageable components (micronuclei, multinuclear, GPCR (G protein receptor) aggregates, etc.) in the same cell can be reliably distinguished from other cells and accurately analyzed. In addition to the cytoplasm, the cell membrane may be specifically labeled. In this case, the cell can be recognized without affecting the analysis of the components in the cytoplasm as in Example 3 described above. It becomes possible. In addition, if it is a part other than the distribution region or part of the biologically active ingredient to be examined, a fluorescent label (dye or fusion protein) is used for the label, so that only cell recognition is performed reliably. However, in this case as well, it is preferable not to cause an indirect adverse effect by appropriately setting the fluorescence label or excitation light so that the fluorescence intensity is minimized as much as possible.

  Examples of bioluminescence (or chemiluminescence) that can be used in the present invention include cells or tissues into which DNA containing a luciferase gene has been introduced as a reporter gene linked downstream of a promoter of a specific gene region of interest. . By using a cell or tissue in which luciferase is expressed as a reporter, it is possible to detect changes in transcription over time by detecting luciferase activity at a desired expression site.

  A preferred embodiment is a vertebrate cell or tissue in which the introduced luminescent gene is expressed in a rhythm in peripheral tissues. Peripheral tissues include, but are not limited to, liver, lung, and skeletal muscle. These peripheral tissues are reported to have a circadian rhythm with a phase difference of 7-12 hours. The delay pattern of circadian rhythm is thought to reflect the normal coordination of the biological rhythm of complex mammals composed of multiple organs.

  According to this, the information analyzed according to the present invention is intended to elucidate the mechanism of jet lag or sleep disorder related to circadian rhythm, and to screen and test compounds useful for the treatment of circadian rhythm disorder. It can be said that it is useful for developing a mammal model to be used as

  Moreover, various tests or screenings can be performed by using a transformant or a transgenic mammal containing the DNA of the present invention that expresses a reporter gene. By detecting reporter gene expression in these tissues or cells under a variety of arbitrary conditions, it is possible to assess or screen for the effects of stimuli or compounds that modulate reporter gene expression . Stimulation includes temperature, light, exercise, and other shocks. There are no restrictions on the compounds used. In particular, the present invention relates to a compound that modifies the expression induced by the promoter of a clock gene (for example, Period 1) introduced into the transformant or transgenic mammal of the present invention, and the transformant or transgenic mammal. It is applicable to the method of using or testing.

Examples of the test or screening method of the present invention include the following methods.
Method (1): A method for testing or screening a compound having an activity of altering the expression of a transgene in the transformant of the present invention, comprising: (a) treating the transformant with the compound; b) measuring the expression of the transgene in the treated transformant.
Method (2): A method for testing or screening a compound having an activity of altering transgene expression in a mammal of the present invention, comprising: (a) treating the mammal with the compound; and (b) Measuring the expression of the transgene in the treated mammal.

The method of the present invention is useful for screening compounds that modulate the expression of Period 1 gene. The method is also useful for screening pharmaceuticals for circadian rhythm disorders. Therefore, in addition to the above methods (1) and (2), the following screening methods are also possible according to the present invention.
Method (3): A method for testing or screening a pharmaceutical agent useful for the treatment of circadian rhythm sleep disorder, wherein (a) treating the transformant or transgenic non-human mammal of the present invention with the pharmaceutical agent; And (b) measuring the expression of a reporter gene in the treated transformant or mammal.

  There is no particular limitation on the compound used in the test or screening method of the present invention. Examples include inorganic compounds, organic compounds, peptides, proteins, natural or synthetic low molecular compounds, natural or synthetic high molecular compounds, tissue or cell extracts, microbial culture supernatants, plants or marine organisms. Natural ingredients derived from, etc. are included, but are not limited thereto. Expression products such as gene libraries or cDNA expression libraries may be used. There is no particular limitation on the method of treatment with the compound. In vitro treatment may be performed, for example, by adding the compound to the culture medium and contacting the cell with the compound, or introducing the compound into the cell using a microinjection or transfection reagent. Methods of treatment in vivo include intraarterial injection, intravenous injection, subcutaneous injection, or intraperitoneal injection; oral administration, enteral administration, intramuscular administration, or intranasal administration; ocular administration; injection or catheterization. Methods known to those skilled in the art, such as intracerebral administration, intraventricular administration, or peripheral organ administration, are included. The compound is suitably administered as a composition. For example, it can be mixed with water, saline, buffers, salts, stabilizers, preservatives, suspending agents and the like.

  Reporter gene expression can be measured while the mammal or cell is alive, or can be measured after solubilizing the cell. For example, to measure the expression of a luciferase gene in living tissue, Yamazaki, S. et al. (Science, 2000, 288) incorporated by reference as described herein with reference to photomultiplier tubes. 682-685), it is possible to measure bioluminescence continuously. The luciferase activity in solubilized tissue or cells can be measured using, for example, a Luciferase Reporter Assay System (Promega). Reporter gene expression can be measured temporally or spatially. Expression can also be analyzed by detecting the phase, amplitude, and / or period of the expression rhythm. The method of the present invention makes it possible to evaluate the immediate or long-term effects (including phase changes) of a compound. If these expressions are altered by administration of a compound, the compound becomes a drug candidate that regulates the expression of the Period 1 gene. Such compounds are expected to be applied as pharmaceuticals against various circadian rhythm disorders including sleep disorders. For example, an agent that resets or initiates a reporter gene expression oscillation would be expected to reverse or advance the pacemaker phase. Thus, these agents can be used to direct a desynchronized expression pattern to normal synchronization. The pharmaceutical product screened by the present invention is administered to the transgenic mammal of the present invention induced to become a circadian rhythm disorder model in order to evaluate the therapeutic effect of the drug.

  When detecting gene expression in a transgenic mammal, the organ to be measured is not particularly limited, and this includes the central nervous system (CNS) and the peripheral nervous system (PNS) including the hypothalamic SCN, liver, lung, And other peripheral tissues including but not limited to skeletal muscle. The system disclosed in the present invention is useful for assessing the phase relationship and synchronization mechanism of Period 1 expression in SCN and peripheral tissues.

  The system of the present invention can be used to identify a number of factors presumed to modulate Period 1 expression. Once new in vivo factors and genes related to circadian rhythm are identified using the system of the present invention, the in vivo oscillations of expression of these factors and genes can be assessed. Thereby, it is possible to isolate factors that control the vibration phase of SCN and peripheral tissues. These are considered to be novel genes and proteins involved in circadian rhythm, and by using these as targets, it is considered that screening for new drugs becomes possible. Such screening can be done both in vivo and in vitro.

Specifically, the in vivo screening method using the transgenic mammal of the present invention is achieved by the method (4) including the following steps.
Method (4): (a) administering a compound to a transgenic mammal whose circadian rhythm has already been determined; (b) periodically detecting the expression level of the reporter gene in the transgenic mammal, and expressing the rhythm (C) comparing the expression rhythm of the reporter gene after administration of the compound with that before administration; and (d) selecting a compound that alters the phase, period, or amplitude of the expression rhythm.

  The expression rhythm of the reporter gene can be determined by a method of detecting the expression rhythm of the reporter gene in the living animal body; a method of continuously observing expression fluctuations by culturing the excised tissue; or an extract of animal tissue It can be detected by a method that is prepared periodically and detects the expression level at each time point. For example, luciferin is administered to a transgenic animal at an appropriate timing by a suitable method (eg, intravenous injection, intraperitoneal administration, intraventricular administration, etc.). Subsequently, the animal is anesthetized, and the luciferase luminescence is measured by a CCD camera to determine the expression site and expression level of the reporter gene. This measurement is performed several times every few hours to confirm the expression rhythm of individual animals (Sweeney TJ et al., “Visualizing the kinetics of tumor-cell clearance in living animals ) ", PNAS, 1999, 96, 12044-12049; and Contag PR et al.," Bioluminescent indicators in living mammals ", Nature Medicine, 1998, 4, 245-247. ).

As described above, a novel drug can be screened in vitro using the present invention. Such an in vitro screening method can be achieved by the method (5) including the following steps.
Method (5): (a) culturing a tissue or cell derived from the transformant of the present invention or the transgenic mammal of the present invention; (b) compound of the transformant or tissue or cell over an appropriate period of time; (C) a step of periodically detecting the expression level of the reporter gene; and (d) the expression rhythm (phase, cycle, and amplitude) of the reporter gene after the treatment of (b). Selecting a compound to be modified.

  As used herein, the tissue or cell to be imaged of the present invention may be a primary culture or an established cell line cell. Although there is no restriction | limiting in the tissue, cell, etc. which are used by this specification, SCN in a vertebrate, a hypothalamic margin cell, a peripheral nerve, etc. are preferable. The treatment with the compound may be performed, for example, by immersing tissues, cells, and the like for a certain period in a solvent to which the compound has been added. When measuring the change in the expression rhythm of the reporter gene, comparison may be performed using the same tissue or cell in which the expression rhythm has been determined in advance, or a control tissue or cell that has not been treated with the compound.

  In the in vivo and in vitro screening methods described above, stimulation treatment such as light stimulation may be performed together with administration or treatment of the compound.

  A compound identified by the test or screening method of the present invention can be used as a pharmaceutical agent for a desired circadian rhythm disease or disorder. These agents can be formulated as pharmaceutical compositions by appropriately combining with pharmaceutically acceptable carriers, solutes and solvents. The drug can be applied to diseases or disorders such as jet lag syndrome, sleep disorder due to shift work, sleep phase regression syndrome, and irregular sleep-wake disorder.

Claims (12)

In a method for monitoring a living sample for a long time by luminescence based on photoprotein, the sample having a thickness of 300 μm or more into which a photoprotein is introduced according to an item to be monitored under a processing condition that does not impair biological activity is used as a substrate. Chemically induced by component, continuously inducing and imaging the emission during the desired monitoring period, with imaging volumes less than 300 μm from the surface of the sample and for multiple different times at the single cell level long-term monitoring method according to the light emitting protein obtain an optical image, said plurality of optical image monitor display, characterized by. The method of claim 1, monitoring method characterized by the test sample is woven bio sets of wall thickness. 3. The monitoring method according to claim 2, wherein the living tissue is an animal or plant organ. 4. The monitoring method according to claim 3, wherein the organ is an embryo, brain, hand, foot, hair root, leaf, flower stem or root hair. 5. The method according to claim 3, wherein the animal is a zebrafish, a mouse or a Xenopus laevis, and the plant is an Arabidopsis thaliana. 4. The monitoring method according to claim 3, wherein the biological sample is a biological material for regenerative medicine. 4. The monitoring method according to claim 3, wherein the biological sample is a transgenic non-human mammal. 2. The monitoring method according to claim 1 , wherein the sample includes a group of cells arranged in a two-dimensional substantially constant state. 9. The monitoring method according to claim 8 , wherein the cell group has a density such that the cells do not overlap on the optical path for imaging. According to claim 8 or 9, monitoring method, characterized in that said group of cells were prepared in the slice sections or sheet-like cells. The method according to any one of claims 1 to 10 , wherein the substrate is firefly luciferin or coelenterazine. 12. The method according to claim 1, wherein the square value of (NA ÷ β) expressed by numerical aperture (NA) and projection magnification (β) is 0.01 or more. A monitoring method, which is performed using an optical condition including an objective lens.

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