CN106978444B - Method for introducing nucleic acid into cell - Google Patents
Method for introducing nucleic acid into cell Download PDFInfo
- Publication number
- CN106978444B CN106978444B CN201610028936.4A CN201610028936A CN106978444B CN 106978444 B CN106978444 B CN 106978444B CN 201610028936 A CN201610028936 A CN 201610028936A CN 106978444 B CN106978444 B CN 106978444B
- Authority
- CN
- China
- Prior art keywords
- cell
- nucleic acid
- exogenous nucleic
- cells
- acid
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
Landscapes
- Genetics & Genomics (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Organic Chemistry (AREA)
- Biotechnology (AREA)
- General Engineering & Computer Science (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Biomedical Technology (AREA)
- Microbiology (AREA)
- Plant Pathology (AREA)
- Molecular Biology (AREA)
- Physics & Mathematics (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Biophysics (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention provides a method for quickly, simply and efficiently introducing nucleic acid into cells. Specifically, a method for introducing a nucleic acid into a cell by adjusting the cell culture environment, changing the state of the cell, and the like is provided. According to the method, the eukaryotic cell culture environment is adjusted to be acidic, so that the nucleic acid substance can enter the eukaryotic cell more efficiently. The method is convenient and efficient to operate, does not need to add an exogenous vector, and has low toxicity to cells, high transfection efficiency and more stable introduced nucleic acid molecules.
Description
Technical Field
The present invention relates to the field of molecular biology, more specifically to a method for introducing nucleic acids into eukaryotic cells.
Background
Research based on eukaryotic cultured cells is the basis of modern biology. Since the cultured eukaryotic cells not only possess complete replication and gene expression machinery, signal pathways and metabolic pathways, but also avoid the high complexity of organisms to a great extent, the cultured eukaryotic cells are usually used as a model system for research before the research of eukaryotes.
Introduction of nucleic acids (DNA, RNA) into cultured cells of eukaryotes (including mammals, plants, insects, etc.) is a key technology in molecular biology, by which humans can express cloned genes in cultured cells of eukaryotes and easily quantify the amount of expression, and protein-protein and protein-nucleic acid interactions can be analyzed. The expression of certain genes can also be shielded (knock-out) or reduced (knock-down) by introducing antisense DNA, SiRNA (small interfering RNA) into cultured cells of eukaryotes. In a broader sense, nucleic acid introduction allows the study and regulation of almost all intracellular activities such as nucleic acid replication, gene expression, signal transduction, and metabolism. Such work is of great significance, and has played a great role in aspects such as production and preparation of active proteins, research on the structure and biochemical characteristics of expressed proteins, research on the regulation and control mechanism of gene expression, and the like. It is considered that the technique of introducing nucleic acid into cultured cells of eukaryotes is one of the core techniques of the whole molecular biology.
Researchers have developed various techniques for introducing nucleic acids, most commonly by calcium phosphate precipitation or liposome-mediated methods, but all have drawbacks in practical applications. In the calcium phosphate precipitation method, the calcium phosphate-nucleic acid complex precipitate can adhere to the surface of the cell membrane and enter the cytoplasm by endocytosis. The size and quality of the precipitated particles in this method are critical to the results, are severely affected by various external conditions, and have poor reproducibility. The liposome-mediated method may be a method in which nucleic acid is encapsulated with a lipid membrane by a neutral lipid (lipid), and introduced into a cell membrane via the lipid membrane or a method in which positively charged Cationic liposomes (Cationic liposomes) are used, which can form a nucleic acid-Cationic liposome complex with negatively charged nucleic acid, adsorb to the surface of the negatively charged cell membrane, and be introduced into cultured cells by endocytosis. The method has high toxicity to cells and unstable effect on different cells. Other methods include electroporation, microinjection, particle gun method, etc., but these methods require expensive equipment and relatively complicated techniques, and cannot be widely popularized.
Therefore, there is an urgent need in the art to develop a method that has little effect on cells and can introduce nucleic acids into cells rapidly, simply, and efficiently.
Disclosure of Invention
The present invention aims to provide a method for introducing a nucleic acid into a cell rapidly, simply and efficiently
In a first aspect of the invention, there is provided an in vitro method for introducing an exogenous nucleic acid into a cell, comprising the steps of:
(a) providing an exogenous nucleic acid and a cell to be transfected;
(b) contacting the cell with the exogenous nucleic acid under acidic pH conditions, thereby introducing a portion of the exogenous nucleic acid into the cell.
In another preferred embodiment, the pH is at pH2.5-6.0, preferably 2.8-5.5, more preferably 2.9-4.5, most preferably 3.0-4.0.
In another preferred embodiment, in step (b), the adjustment is performed with an acid, thereby obtaining said acidic pH condition.
In another preferred embodiment, the acid comprises an organic acid, an inorganic acid, or a combination thereof.
In another preferred embodiment, the acid is selected from the group consisting of: hydrochloric acid, phosphoric acid, sulfuric acid, or a combination thereof.
In another preferred embodiment, the cell is contacted with the exogenous nucleic acid for a period of time ranging from 0.5 minutes to 24 hours, preferably from 1 minute to 6 hours, more preferably from 5 minutes to 2 hours, and most preferably from 0.25 to 1 hour.
In another preferred embodiment, the cell is contacted with the exogenous nucleic acid at a temperature of 4-50 deg.C, preferably 15-45 deg.C, more preferably 25-40 deg.C, and most preferably 35-39 deg.C.
In another preferred embodiment, the exogenous nucleic acid is selected from the group consisting of: DNA, RNA, and DNA/RNA hybrid nucleic acids.
In another preferred embodiment, the exogenous nucleic acid is selected from the group consisting of: small RNA molecules, and plasmids.
In another preferred embodiment, the small RNA molecule is selected from the group consisting of: siRNA, miRNA, and shRNA.
In another preferred embodiment, the exogenous nucleic acid includes single-stranded nucleic acid and double-stranded nucleic acid.
In another preferred embodiment, the exogenous nucleic acid has a length of 10-8000bp, preferably 15-5000bp, more preferably 18-1000bp, most preferably 20-100 bp.
In another preferred embodiment, the concentration of the exogenous nucleic acid is 0.1 to 1000pmol/mL, preferably 0.5 to 200pmol/mL, more preferably 1 to 100pmol/mL, most preferably 10 to 50 pmol/mL.
In another preferred embodiment, the cell includes prokaryotic cells and eukaryotic cells.
In another preferred embodiment, the cell is a mammalian cell.
In another preferred embodiment, the cell is a somatic cell, a pluripotent stem cell, and/or a germ cell.
In another preferred embodiment, the method is a non-therapeutic, non-diagnostic method.
In a second aspect of the present invention, there is provided an in vitro transfection system for introducing an exogenous nucleic acid into a cell, comprising:
(i) an aqueous system having an acidic pH;
(ii) an exogenous nucleic acid; and
(iii) cells to be transfected.
In a third aspect of the present invention, there is provided an in vitro method for introducing an exogenous nucleic acid into a cell, comprising the steps of:
(a) providing an exogenous nucleic acid and a cell to be transfected;
(b1) treating the cells under acidic pH conditions, thereby obtaining acidified treated cells;
(b2) contacting the acidified cell with the exogenous nucleic acid, thereby introducing a portion of the exogenous nucleic acid into the cell.
It is to be understood that within the scope of the present invention, the above-described features of the present invention and those specifically described below (e.g., in the examples) may be combined with each other to form new or preferred embodiments. Not to be reiterated herein, but to the extent of space.
Drawings
FIG. 1 shows the relative amount of osa-156a in wild type 293T cells treated at different pH.
FIG. 2 shows Lipofectamine TM2000 and acid induced transfection FAM siRNA fluorescence results are shown.
FIG. 3 shows a graphical representation of the relative transfection ratios of two pH media after transformation into pCMV-SPORT- β -gal plasmid in HepG2 cells.
FIG. 4 shows a bar graph of the transformation efficiency of cells treated with hydrochloric acid, phosphoric acid, and sulfuric acid.
FIG. 5 shows a bar graph of the transformation efficiency of cells treated at different pH values.
FIG. 6 shows a bar graph of transformation efficiency for different concentrations of nucleic acid transfection.
Detailed Description
The present inventors have made extensive and intensive studies and have unexpectedly found a method for introducing a nucleic acid into a cell rapidly, simply and efficiently. Experiments show that nucleic acid substances can enter eukaryotic cells more efficiently by adjusting the culture environment of the eukaryotic cells to be acidic. The method is convenient and efficient to operate, does not need to add an exogenous vector, and has low toxicity to cells, high transfection efficiency and more stable introduced nucleic acid molecules. On the basis of this, the present invention has been completed.
Term(s) for
Small ribonucleic acid (Small RNA)
In the present invention, the term "small ribonucleic acid (small RNA)" refers to a small fragment of RNA that is twenty-several nucleotides in length; according to the widely accepted classification method proposed by Steven Buckingham in 5 months 2003, small RNA (small RNAs) refers to the portion of non-coding RNA other than transcribed RNA (including ribosomal RNA and transfer RNA), including micro RNA (micrornas), small interfering RNA (short interfering RNAs, siRNA), small nucleolar RNA (snorna), and small nuclear RNA (snrna).
Wherein, the microRNA (miRNA) is a single-stranded microRNA molecule with the length of about 19-23 nucleotides, is positioned in a genome non-coding region, is highly conserved evolutionarily, can regulate gene expression by inhibiting the translation process of a target gene, is closely related to a plurality of normal physiological activities of animals, such as biological ontogeny, tissue differentiation, cell apoptosis, energy metabolism and the like, and is closely related to the occurrence and development of a plurality of diseases. Existing studies have also demonstrated that vegetal mirnas can also enter the body of animals by ingestion and participate in regulatory activities.
Small interfering ribonucleic acid (siRNA) is a double-stranded RNA molecule consisting of more than 20 nucleotides, and can play a role in silencing gene expression by specifically degrading messenger RNA (mRNA) of a target gene. This process is called RNA interference (RNAi). RNA interference is a means of post-transcriptional regulation of genes. sirnas specifically recognize their target genes and recruit protein complexes called silencing complexes (RISC). RISC contains ribonuclease, etc., and can specifically and efficiently inhibit gene expression by targeting cleavage of homologous mRNA. Since the expression of a specific gene can be specifically knocked out or turned off using the RNA interference technique, the technique has been widely used in the field of biomedical experimental research and treatment of various diseases.
The main advantages of the invention include:
(a) the method improves the efficiency of transfecting cells by nucleic acid substances by adjusting the cell culture environment to be acidic.
(b) The method of the invention has the advantages of low toxicity, high transfection efficiency and high stability of the introduced nucleic acid molecules.
(c) The method is convenient and efficient to operate, and greatly simplifies the time and steps of laboratory operation.
The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. The experimental procedures, in which specific conditions are not noted in the following examples, are generally carried out under conventional conditions or conditions recommended by the manufacturers. Unless otherwise indicated, percentages and parts are by weight.
Example 1
Introduction of osa-miRNA156a into wild-type 293T cells
In this example, the experimental method is as follows:
1) PDL (PDL) envelope
250mL of 0.01% polylysine was prepared:
containers and handling equipment were sterilized and polylysine was purchased from sigma.
A small amount of PBS was pipetted into a small plastic bottle of polylysine, and then the dissolved polylysine was added to about 200mL of PBS.
Finally adding PBS to 250mL, filtering, sterilizing, subpackaging in small bottles, storing at 4 ℃ for later use, and storing at-20 ℃ for a long time.
2) Floor board
Adding 0.01% polylysine into each well with a 12-well plate, 3-4 drops per well, covering the 12-well plate, placing at 37 deg.C and 5% CO2And incubating for 2-4h in the incubator.
After the incubation was completed, the 12-well plate was removed and polylysine was aspirated out with a pipette. Change the pipet, add PBS, 3-4 drops per hole, wash three times. The above steps were repeated three times.
The 12-well plate is placed in an incubator for use.
3) Cell culture
The 293T cells were plated into 2 12-well plates
The culture conditions are as follows: DMEM (10% FBS, 1% double antibody), 37 ℃, 5% CO2 culture for 24 h.
4) Preparation of acidic culture solution
DMEM medium (2% FBS, 1% double antibody) used in the experiment was first prepared and its pH was measured to be 7.65, after which the culture was adjusted to pH3.15 with hydrochloric acid for further use.
5) Transfer of nucleic acid
The transferred nucleic acid is rice single-stranded microRNA synthesized by exogenous sources, osa-miR156a (synthesized by Gimera, sequence UGACAGAAGAGAGUGAGCAC (SEQ ID NO.:1)) and rice osa-miR156a has great difference with transferred human 293T cells, so that the interference of the microRNA in animal cells can be effectively avoided, and the possibility of error generation in experimental results is reduced.
osa-miR156a was added to the culture broth at pH7.65 and pH3.15, respectively, to a final concentration of 40 pmol/mL.
Taking out 12-well plate paved with 293T cells from the incubator, removing original culture solution, adding the culture solution with two pH values added with exogenous miRNA into 2 12-well plates, and incubating at 37 deg.C for 0.5 h.
During the incubation period, RNase digest was prepared: RNase A (purchased from Thermo) was added to DMEM medium (2% FBS, 1% double antibody) in a volume ratio of 2. mu.L/mL, i.e. >100 u/mL.
After the incubation time is over, removing the culture solution with two pH values added with exogenous miRNA, washing with PBS once, adding prepared RNase digestive juice, incubating for 1h at 37 ℃, and washing with PBS three times after removal.
Cells were lysed with TRI Regent (purchased from Sigma) and total RNA was extracted as follows:
cracking at room temperature for 5 min; adding 1/5 volumes of chloroform, shaking vigorously, and standing at room temperature for 5 min; 16000g, centrifuging at 4 deg.C for 15 min; carefully sucking out the supernatant, adding 2 times of isopropanol, mixing uniformly, and standing at-20 ℃ for 1h or overnight; 16000g, centrifuging at 4 deg.C for 15 min; discarding the supernatant, adding 1mL of 75% ethanol (prepared by DEPC water) to wash the precipitate, 16000g, centrifuging at 4 ℃ for 15 min; pouring off the supernatant, and adding a proper amount of DEPC water to dissolve the precipitate after the precipitate is dried; OD260/OD280 were calculated to identify total RNA concentration and purity.
6) Detection of results
Quantitatively detecting the miRNA content in the cells by utilizing a qRT-PCR technology:
the real-time fluorescent quantitative PCR technology is that fluorescent group is added into the reaction system of PCR and fluorescent signal accumulation is utilized to monitor the whole PCR process in real time. In view of the length of miRNA 21bp, a gene-specific reverse primer containing the same stem-loop structure (stem-loop structure) is designed for each miRNA, a specific cDNA is obtained by reversion, and finally PCR reaction is carried out.
Preparing the extracted RNA and other reagents required by the reaction into a reverse transcription reaction system, and carrying out the reaction according to the following conditions:
step 1: 30min at 16 DEG C
Step 2: 30min at 42 DEG C
And step 3: 5min at 85 DEG C
And 4, step 4: 4 ℃ forever
After the corresponding cDNA is obtained according to the reaction, the cDNA and other reagents required by the reaction are prepared into a PCR reaction system, the reaction is carried out according to the following conditions to obtain a fluorescence signal value, and the real-time fluorescence quantitative PCR reaction conditions are as follows:
step 1: 5min at 95 DEG C
Step 2: 95 ℃ for 15s
And step 3: 60 ℃ for 1min
Step 2-step 3: 50 cycles
The data is processed using a relative comparison method, also known as the Δ Δ Ct method. Ct is the cycle number at which the reaction reaches the threshold, and Δ Ct ═ Ct sample-Ct internal reference. The expression level of the acid-treated exogenous microRNA relative to the control wild-type plant can be determined by equation 2-ΔCTAnd (4) showing.
The results are as follows:
FIG. 1 shows the relative values of osa-miR156a transferred into cells by two pH culture media, wherein the relative value of a pH7.65 control group is 1, and the result shows that the content of the exogenous microRNA in the cells after the exogenous microRNA of the pH3.15 group is transformed is obviously higher than that of the pH7.65 control group.
Example 2
By Lipofectamine TM2000 and acid-induced transfection of FAM siRNA
In this example, the experimental method is as follows:
1)Lipofectamine TM2000 transfection
cells were plated on a 12-well plate one day before transfection, and 400. mu.L of DMEM medium (without double antibody) containing 10% calf serum was added to each well, and the cell density at transfection was 90%.
Serum-free medium (Opti-MEM I) was replaced 4-6 hours before transfection, and Lipofectamine was used 4-6 hours after culture TM2000 siRNA was transfected into cells, the specific steps were as follows:
100pmol of siRNA was diluted into 45. mu.L of Opti-MEM I and gently mixed.
Mix Lipofectamine gentlyTM2000, then 1. mu.L of the suspension was diluted in 49. mu.L of Opti-MEM I, gently mixed, and incubated at room temperature for 5 minutes.
Mixing the diluted siRNA with Lipofectamine TM2000 mix gently and incubate for 20 minutes at room temperature to form a complex.
Add 100. mu.L of complex to each well and translate the plate back and forth and left and right to mix the complex with the cells.
The medium is changed to a serum-containing medium 4-6 hours after transfection (which can be prolonged appropriately).
2) Acid-induced transfection of FAM siRNA
The method of acid-induced transfection of FAM siRNA was the same as described in example 1.
3) Detection of transfection efficiency
FAM is a green fluorophore with blue excitation, excitation wavelength of 480nm and emission wavelength of 520 nm.
LipofectamineTMThe detection can be carried out 6 hours after the transfection, and the cell treatment process before the detection needs to be protected from light. During detection, the light path can be aligned to a hole without transfection of FMA siRNA, and the focus is adjusted to turn on the exciting light. The observation time should not be too long to avoid quenching the fluorescence.
Successfully transfected cells were visualized for FAM green fluorescence, scattered in the cytoplasm.
The results are as follows:
FIG. 2 is Lipofectamine TM2000 and acid-induced transfection FAM siRNA fluorescence results are shown in the figure, which shows thatThe transfection efficiency of the two groups is equivalent, which indicates that the acid treatment group also has the capability of efficiently transfecting siRNA.
Example 3
Introduction of pCMV-SPORT-beta-gal plasmid into HepG2 cells
PDL coating, plating, etc. procedures were the same as described in example 1, and the introduced pCMV-SPORT-. beta. -gal plasmid was purchased from Life technologies.
The experimental method is as follows:
1) cell culture
HepG2 cells were plated into 2 12-well plates
The culture conditions are as follows: DMEM (10% FBS, 1% double antibody), 37 ℃, 5% CO2 culture for 24 h.
2) Preparation of acidic culture solution
DMEM medium (2% FBS, 1% double antibody) used in the experiment was first prepared and its pH was measured to be 7.65, after which the culture was adjusted to pH3.15 with hydrochloric acid for further use.
3) Transfer of nucleic acid
The transferred nucleic acid is selected from pCMV-SPORT-beta-gal plasmid, the pCMV-SPORT-beta-gal is a vector for detecting transfection efficiency, an escherichia coli beta glucuronidase gene carried by the vector is positioned behind a CMV (cytomegalovirus) promoter for driving high-level transcription of mammalian cells, and SV40 polyadenylation signals at the downstream of a beta glucuronidase sequence guide the correct processing of eukaryotic mRNA.
To the culture medium at pH7.65 and pH3, pCMV-SPORT-. beta. -gal plasmid was added to a final concentration of 40pmol/mL, respectively.
The 12-well plate plated with HepG2 cells was removed from the incubator, and the original culture medium was removed, and the two kinds of culture media with pH values added with pCMV-SPORT-. beta. -gal plasmid were added to 2 12-well plates, respectively, and incubated at 37 ℃ for 0.5 h.
After the incubation time is over, removing the culture solution with two pH values added with pCMV-SPORT-beta-gal plasmid, washing once with PBS, adding the prepared DNase digestive juice, incubating for 1h at 37 ℃, and washing three times with PBS after removing.
4) Detection of results
The monolayer histochemical staining was used to identify beta-glucuronidase, which was determined as follows:
reagent:
phosphate buffer
1mg/mL X-gal
Cell fixing agent: 2% (V/V) formaldehyde, 0.2% (V/V) glutaraldehyde, 1 XPhosphate buffer
And (3) grouping and dyeing: 5mmol/L potassium ferricyanide, 5mmol/L potassium ferrocyanide, 2mmol/L MgCl21 XPhosphate buffer solution
The staining solution was stored at 4 ℃ in the dark and X-gal was added rapidly before cell staining.
Method
1. Transfected cells were washed twice with 2-3mL of phosphate buffer at room temperature.
2.5 mL of cell fixative was added.
3. Cells were washed once with phosphate buffer.
4. 3-5mL of a organized staining solution was added.
Cells were incubated at 5.37 ℃ for 14-24 h.
6. The monolayer of cells was washed several times with phosphate buffer.
7. Cells were covered with a small amount of phosphate buffer and examined under an optical microscope.
Cells expressing the β -glucuronidase vector appeared bright blue, and the transfection ratio was calculated from the relative number of stained and unstained cells.
The results are as follows:
FIG. 3 shows the relative values of transfection ratios after two pH media are transferred into pCMV-SPORT-beta-gal plasmid in HepG2 cell, wherein the relative value of pH7.65 control group is 100%, the result shows that the content of exogenous plasmid in the cell after the exogenous plasmid of pH3 group is transferred is relatively higher than that of pH7.65 control group, and the acid treatment method can produce effect on HepG2 cell.
Example 4
Effect of different acids on conversion efficiency
The experimental procedure was the same as described in example 1, the transformation efficiency was determined by introducing nucleic acid as pCMV-SPORT- β -gal plasmid, adjusting the pH of the medium to 3.15 during the experiment using hydrochloric acid, phosphoric acid and sulfuric acid, respectively, and the other parameters were not changed, and after the transfection, the β -glucuronidase activity was determined according to the procedure of example 3 to determine the transformation efficiency of each group, and the transfection ratio was calculated from the relative number of stained and unstained cells.
The results are shown in fig. 4, and fig. 4 shows a bar chart of the conversion efficiency of hydrochloric acid, phosphoric acid and sulfuric acid, and it can be seen that all three acids can complete the conversion and have higher conversion efficiency, the conversion rates of the three acids have no significant difference, which indicates that the acidic condition is the key of the method of the present invention, and the anions in the acids have no significant influence on the conversion rate. In three experiments, the conversion efficiency of hydrochloric acid is highest, the conversion efficiency of phosphoric acid is lowest, and the conversion efficiency of sulfuric acid is highest.
Example 5
Effect of different pH on conversion efficiency
The experimental procedure was the same as described in example 1, the nucleic acid was introduced as pCMV-port- β -gal plasmid to identify the transformation efficiency, the pH of the medium was adjusted to 3, 3.15, 3.3, 3.45 during the experiment, and the other parameters were not changed, the β -glucuronidase activity was identified according to the procedure of example 3 after the end of transfection to determine the transformation efficiency of each group, and the transfection ratio was calculated from the relative number of stained and unstained cells.
As shown in FIG. 5, FIG. 5 is a bar graph showing the conversion efficiency at pH values of 3, 3.15, 3.3 and 3.45, respectively, and it can be seen that the conversion can be completed at four different pH values and the conversion efficiency is relatively high, wherein the conversion efficiency is relatively highest at pH value of 3.15. The result shows that the method has no strict requirement on the acid pH condition, and has higher conversion rate under a wider acid pH condition.
Example 6
Effect of different transfection concentrations on transformation efficiency
The experimental procedure was the same as described in example 1, the introduction of nucleic acid as pCMV-SPORT- β -gal plasmid to identify the transformation efficiency, the final concentration of the transfected plasmid was adjusted to 35, 40, 45pmol/mL respectively during the experiment, other parameters were unchanged, the β -glucuronidase activity was identified according to the procedure of example 3 after the end of transfection to determine the transformation efficiency of each group, and the transfection ratio was calculated from the relative number of stained and unstained cells.
As shown in FIG. 6, FIG. 6 is a bar graph showing the transformation efficiencies at final concentrations of 35, 40 and 45pmol/mL of the transfected plasmid, indicating that the transformation was completed and the transformation efficiency was high at three different nucleic acid concentrations, of which the highest transformation efficiency was achieved at a nucleic acid concentration of 40 pmol/mL. The results show that the method of the invention has no strict requirements on the concentration of nucleic acid, and can achieve higher conversion rate at lower concentration of nucleic acid.
All documents referred to herein are incorporated by reference into this application as if each were individually incorporated by reference. Furthermore, it should be understood that various changes and modifications of the present invention can be made by those skilled in the art after reading the above teachings of the present invention, and these equivalents also fall within the scope of the present invention as defined by the appended claims.
Claims (17)
1. An in vitro method for introducing exogenous nucleic acid into a cell, comprising the steps of:
(a) providing an exogenous nucleic acid and a cell to be transfected;
(b) contacting said cell with said exogenous nucleic acid under acidic pH conditions, thereby introducing a portion of said exogenous nucleic acid into said cell, said cell being a eukaryotic cell, wherein said eukaryotic cell is a HepG2 cell or a 239T cell; the pH condition is pH 2.9-4.5.
2. The method of claim 1, wherein the pH condition is pH3.0 to 4.0.
3. The method of claim 1 wherein in step (b) said acidic pH condition is achieved by acid adjustment.
4. The method of claim 3, wherein the acid comprises an organic acid, an inorganic acid, or a combination thereof.
5. The method of claim 1, wherein said cell is contacted with said exogenous nucleic acid for a time period ranging from 0.5 minutes to 24 hours.
6. The method of claim 1, wherein the cell is contacted with the exogenous nucleic acid at a temperature of 4 ℃ to 50 ℃.
7. The method of claim 1, wherein said exogenous nucleic acid is selected from the group consisting of: DNA, RNA, and DNA/RNA hybrid nucleic acids.
8. The method of claim 1, wherein said exogenous nucleic acid is selected from the group consisting of: small RNA molecules, and plasmids.
9. The method of claim 8, wherein the small RNA molecule is selected from the group consisting of: siRNA, miRNA, and shRNA.
10. The method of claim 1, wherein the exogenous nucleic acid is 10-8000bp in length.
11. The method of claim 1, wherein the exogenous nucleic acid is 15-5000bp in length.
12. The method of claim 1, wherein the exogenous nucleic acid is present at a concentration of 0.1 to 1000 pmol/mL.
13. The method of claim 1, wherein the exogenous nucleic acid is present at a concentration of 0.5 to 200 pmol/mL.
14. The method of claim 1, wherein the cell comprises a prokaryotic cell and a eukaryotic cell.
15. The method of claim 1, wherein said method is a non-therapeutic, non-diagnostic method.
16. An in vitro transfection system for introducing exogenous nucleic acid into a cell, comprising:
(i) an aqueous system having an acidic pH, wherein the pH condition is pH 2.9-4.5;
(ii) an exogenous nucleic acid; and
(iii) the cell to be transfected is a eukaryotic cell, wherein the eukaryotic cell is a HepG2 cell or a 239T cell.
17. An in vitro method for introducing exogenous nucleic acid into a cell, comprising the steps of:
(a) providing an exogenous nucleic acid and a cell to be transfected;
(b1) treating the cells under acidic pH conditions, thereby obtaining acidified treated cells;
(b2) contacting the acidified cell with the exogenous nucleic acid, thereby introducing a portion of the exogenous nucleic acid into the cell; the cell is a eukaryotic cell, wherein the eukaryotic cell is a HepG2 cell or a 239T cell, and the pH condition is pH 2.9-4.5.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610028936.4A CN106978444B (en) | 2016-01-15 | 2016-01-15 | Method for introducing nucleic acid into cell |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610028936.4A CN106978444B (en) | 2016-01-15 | 2016-01-15 | Method for introducing nucleic acid into cell |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN106978444A CN106978444A (en) | 2017-07-25 |
| CN106978444B true CN106978444B (en) | 2021-12-17 |
Family
ID=59340551
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201610028936.4A Active CN106978444B (en) | 2016-01-15 | 2016-01-15 | Method for introducing nucleic acid into cell |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN106978444B (en) |
Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1999029839A1 (en) * | 1997-12-12 | 1999-06-17 | Expression Genetics, Inc. | Grafted copolymers as gene carriers |
| WO2002077264A2 (en) * | 2001-03-22 | 2002-10-03 | Whitehead Institute For Biomedical Research | Arrayed transfection method and uses related thereto |
| CN1479632A (en) * | 2000-10-09 | 2004-03-03 | 拜尔公司 | Complexes for transferring necleic acids into cells |
| CN1655826A (en) * | 2002-05-03 | 2005-08-17 | Fmc生物聚合物联合股份有限公司 | Non-viral gene delivery system |
| CN101285074A (en) * | 2007-04-10 | 2008-10-15 | 苏州市长三角系统生物交叉科学研究院有限公司 | Method for introducing nucleic acid into eucaryote cultured cells |
| CN105175442A (en) * | 2014-06-23 | 2015-12-23 | 西安市中心血站（陕西省血液中心） | Cationic liposome for transfection reagents, preparation and application thereof |
-
2016
- 2016-01-15 CN CN201610028936.4A patent/CN106978444B/en active Active
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1999029839A1 (en) * | 1997-12-12 | 1999-06-17 | Expression Genetics, Inc. | Grafted copolymers as gene carriers |
| CN1479632A (en) * | 2000-10-09 | 2004-03-03 | 拜尔公司 | Complexes for transferring necleic acids into cells |
| WO2002077264A2 (en) * | 2001-03-22 | 2002-10-03 | Whitehead Institute For Biomedical Research | Arrayed transfection method and uses related thereto |
| CN1655826A (en) * | 2002-05-03 | 2005-08-17 | Fmc生物聚合物联合股份有限公司 | Non-viral gene delivery system |
| CN101285074A (en) * | 2007-04-10 | 2008-10-15 | 苏州市长三角系统生物交叉科学研究院有限公司 | Method for introducing nucleic acid into eucaryote cultured cells |
| CN105175442A (en) * | 2014-06-23 | 2015-12-23 | 西安市中心血站（陕西省血液中心） | Cationic liposome for transfection reagents, preparation and application thereof |
Non-Patent Citations (8)
| Title |
|---|
| 外源基因转染真核细胞技术的研究进展;余祖华等;《安徽农业科学》;20080720(第21期);全文 * |
| 根际pH对水稻细胞膜质子泵基因表达的影响;沈静等;《中国水稻科学》;20090710(第04期);全文 * |
| 花背蟾蜍蝌蚪皮肤在类坏死条件下超微结构的变化~*;仝允栩等;《实验生物学报》;19841215(第04期);全文 * |
| 酸对人正常食管粘膜上皮细胞增殖和ERK表达水平的影响;姜志茹等;《南方医科大学学报》;20060828(第08期);全文 * |
| 酸对角膜上皮细胞损害的研究;张泺等;《眼外伤职业眼病杂志(附眼科手术)》;19921231;全文 * |
| 酸敏感离子通道基因重组质粒的构建、表达及其活性测定;苏敬敬等;《神经解剖学杂志》;20091130(第06期);全文 * |
| 酸雨对勋章菊保护酶活性及叶绿素荧光参数的影响;侯维等;《核农学报》;20130727(第07期);全文 * |
| 非病毒载体介导的siRNA细胞内导入机理研究;刘玉洁等;《科学通报》;20140120(第02期);第189页右栏 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN106978444A (en) | 2017-07-25 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Lin et al. | miR-199a*, a bone morphogenic protein 2-responsive microRNA, regulates chondrogenesis via direct targeting to Smad1 | |
| Lee et al. | Expression of versican 3′-untranslated region modulates endogenous microRNA functions | |
| KR20100024407A (en) | Single-stranded cyclic rna, and method for production thereof | |
| CN101755049B (en) | Primary micro RNA expression cassette | |
| KR20160145814A (en) | miRNA enhancing cell productivity | |
| AU2002354121A1 (en) | siRNA Expression System and Method for Producing Functional Gene Knockdown Cell Using the Same | |
| Takabatake et al. | Chemically modified siRNA prolonged RNA interference in renal disease | |
| CN115141804B (en) | Neuron transfection method based on fat exosomes and application thereof | |
| JP2023507181A (en) | Nucleic acid constructs for delivering polynucleotides to exosomes | |
| US20060088837A1 (en) | Expression system for stem-loop rna molecule having rnai effect | |
| Gopalakrishnan et al. | siRNA and DNA transfer to cultured cells | |
| CN106978444B (en) | Method for introducing nucleic acid into cell | |
| Li et al. | Gene silencing in Xenopus laevis by DNA vector-based RNA interference and transgenesis | |
| JP4762897B2 (en) | siRNA expression system | |
| Fricke et al. | Targeted RNA knockdown by crRNA guided Csm in zebrafish | |
| CN118516352A (en) | Novel shRNA expression vector, and preparation and application thereof | |
| Scherr et al. | Knock-down of gene expression in hematopoietic cells | |
| Yuan et al. | Mammalian Pol III promoter H1 can transcribe shRNA inducing RNAi in chicken cells | |
| CN110218701B (en) | Cell model for screening miRNA (micro ribonucleic acid) regulating agent and application | |
| CN103103189A (en) | Novel method for overexpression of single MicroRNA (Micro Ribonucleic Acid) mature body sequence | |
| Berger et al. | Inhibition of micro-RNA-induced RNA silencing by 2′-O-methyl oligonucleotides in Drosophila S2 cells | |
| WO2023074873A1 (en) | Cell purification method | |
| García-Sánchez et al. | Gene Silencing Delivery Methods: Lipid-Mediated and Electroporation Transfection Protocols | |
| CN106591305A (en) | siRNA for inhibition of microorganism drug resistance and application of same | |
| Lan et al. | Disease modeling by gene targeting using microRNAs |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |