CN104293831A - Method for establishing hypertension mouse model and application of hypertension mouse model - Google Patents
Method for establishing hypertension mouse model and application of hypertension mouse model Download PDFInfo
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- CN104293831A CN104293831A CN201410510115.5A CN201410510115A CN104293831A CN 104293831 A CN104293831 A CN 104293831A CN 201410510115 A CN201410510115 A CN 201410510115A CN 104293831 A CN104293831 A CN 104293831A
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Abstract
The invention provides a method for establishing a hypertension mouse model and an application of the hypertension mouse model. The method is based on a CRISPR/Cas9 gene knockout technique, and comprises the following steps: 1, establishing an STK39 gene knockout mouse model; 2, identifying the hypertension mouse model; and 3, performing relevant phenotypic analysis on the hypertension mouse model. Based on the CRISPR/Cas9 gene knockout technique, the invention provides a method for establishing the hypertension mouse model, and compared with the prior art, the technical scheme is a method which is convenient and rapid, short in time and good in effect.
Description
Technical field
The present invention relates to a kind of method setting up hypertension mouse model and uses thereof, particularly relate to a kind of based on CRISPR/Cas9 gene Knockout method setting up hypertension mouse model and uses thereof.
Background technology
Mouse disease model serves keying action in research human diseases pathogenesis and drug screening.There is the important diseases model that huge advantage can become research and drug screening probing in human nerve's degenerative disease, neuropsychiatric disease and human cognitive function, neural circuitry etc.
Gene knockout is the Protocols in Molecular Biology complicated based on mouse embryo stem cell DNA homology restructuring one of principle grown up the eighties in last century, also " gene targeting " technology is become, over more than 20 year, utilize this technique construction several thousands genes mutant mice model, these genetic engineering mice not only leap for biological research brings, and also in the development and medicament research and development of medical science, have played vital effect.Just because of this, three the U.S. and Britain scientists of invention technique jointly obtain Nobel's Prize in Physiology/Medicine in 2007.Although then this traditional gene knockout method can be rated as classics, because complicated, the consuming time length of flow process, expense are large, in the past few years enjoy concern and the challenge of scientific circles, everybody is in a kind of quicker, more economical and novel method that suitability is strong of searching.
Being used for studying hypertension animal model at present mainly contains following several: 1, chronic experi Type of Hypertension; 2, Neuron specific emelase; 3, nephrogenic hypertension; 4, endocrine hypertension.These methods setting up hypertension model mostly will through exogenous drugs or other method process, or even by operation means, can not the essential hypertension of simulating human completely, and length consuming time, cost is high, and genetic hypertension model farthest can simulate idiopathic hypertension clinically, also high being conducive to studies hypertensive pathogenesis, the design of medicine target spot.In addition, there is not the difference that operation technique or drug dose cause between essential hypertension model subjects, its background height is consistent, can as the useful tool of new medicament screen.
CRISPR/Cas9 gene Knockout involved by the present invention is the 4th kind of method that can be used for fixing a point to build clpp gene deratization animal after the technology such as Zinc finger nuclease, ES cell targeting and TALEN, to modify show huge potentiality in orientation to gene.The present invention is based on the construction process that CRISPR/Cas9 gene Knockout provides a kind of hypertension mouse model, compared with prior art, technical solution of the present invention is a kind of convenient, consuming time short, effective method.
Summary of the invention
For defect of the prior art, the object of this invention is to provide a kind of method setting up hypertension mouse model and uses thereof, especially a kind of based on CRISPR/Cas9 gene Knockout method setting up hypertension mouse model and uses thereof.
The present invention is achieved by the following technical solutions:
The invention provides a kind of method setting up hypertension mouse model, be the method setting up hypertension mouse model based on CRISPR/Cas9 gene Knockout, described method comprises the steps:
Step one, set up STK39 gene knock-out mice model;
The qualification of step 2, hypertension mouse animal model;
The Relevant phenotype analysis of step 3, hypertension mouse animal model.
Preferably, in step one, describedly set up STK39 gene knock-out mice model, specifically comprise the following steps:
The structure of A, CRISPR targeting modification genophore;
B, mouse induced ovulation and in vitro fertilization;
The microinjection of C, mouse fertilized egg;
D, zygote vitro culture, implantation acceptor and target gene modify the cultivation of animal.
Preferably, in step 2, the qualification of described hypertension mouse animal model comprises:
(1) cervical dislocation sacrifices mouse, extracts the liver of F1 generation chimeric mice and wild control mice, skeletal muscle, fat, kidney and spinal cord total serum IgE; Reverse transcription becomes cDNA; Amplifying target genes, compares the STK39 gene expression difference of heterozygote and wild control mice;
(2) CRISPR target site is carried out to the genotype of DNA sequencing qualification mouse model;
(3) real-time quantitative PCR is utilized to detect the change of STK39 gene mRNA levels in mouse cell;
(4) disappearance of STK39 albumen in mouse cell is detected by immunofluorescence dyeing.
Preferably, in step 3, the Relevant phenotype analysis of described hypertension mouse animal model comprises:
1) Mouse tail artery pressure is measured;
2) by the heart function of small animal imaging systems axiol-ogy mouse;
3) concentration of feritin, Angiotensin, aldosterone in mice serum is detected by ELISA kit;
4) shrink tension of mouse resistance vessel is measured with muscular tension instrument;
5) with myosin Phosphorylation of regulatory light chain in urea glycerin cement electrophoretic analysis mouse resistance vessel;
6) tail arterial blood pressure of mouse is detected after tail vein injection hypertension drug.
Compared with prior art, the present invention has following beneficial effect:
(1) the present invention adopts CRISPR/Cas9 gene Knockout, the target accuracy that tool is higher; Simultaneously cocoa realizes multidigit point and knocks out simultaneously;
(3) cycle obtaining mutant mice is short, only needs 2 months;
(4) present method limits without species, also can carry out operating at the such as animal body such as rat thus obtain corresponding mutant mice;
(5) with the mouse that the inventive method builds, there is the reproductive tract transfer ability being significantly higher than traditional method.
Accompanying drawing explanation
Fig. 1 is that STK39 knocks out the pressure value with control group mice;
Fig. 2 is that STK39 knocks out the shrink tension change of rear blood vessel under various stimulation, comprising 124mM Klorvess Liquid, 10uM norepinephrine (NE), 0.1uM endothelin (ET1), 10uM Angiotensin (AngII), 1uM vassopressin (Vasopressin), 0.1uM thromboxane analogue (U46619);
Fig. 3 is the concentration of feritin (A) during STK39 that ELISA detects knocks out with control group mice serum, Angiotensin (B), aldosterone (C).
Fig. 4 is that STK39 knocks out the cardiac functional parameter with control group mice;
Fig. 5 detects depressor Composition analyzed example for utilizing STK39 knock-out mice;
Wherein, A be perindopril tablets with 1.2ug/g Mouse Weight tail vein injection, continuous five days injection during mouse systolic pressure change; B be amlodipine besylate tablets with 1.5ug/g Mouse Weight tail vein injection, continuous five days injection during mouse systolic pressure change.
Embodiment
Below in conjunction with specific embodiment, the present invention is described in detail.Following examples will contribute to those skilled in the art and understand the present invention further, but not limit the present invention in any form.It should be pointed out that to those skilled in the art, without departing from the inventive concept of the premise, some distortion and improvement can also be made.These all belong to protection scope of the present invention.
embodiment 1
The present embodiment relates to a kind of method setting up hypertension mouse model, and be the method setting up hypertension mouse model based on CRISPR/Cas9 gene Knockout, described method comprises the steps:
Step one, set up STK39 gene knock-out mice model;
The qualification of step 2, hypertension mouse animal model and Relevant phenotype analysis;
The energy metabolism of step 3, hypertension mouse animal model, Regular Insulin, hyperglycemic-glycogenolytic factor evaluation.
In step one, describedly set up STK39 gene knock-out mice model, specifically comprise the following steps:
The structure of A, CRISPR targeting modification genophore;
B, mouse induced ovulation and in vitro fertilization;
The microinjection of C, mouse fertilized egg;
D, zygote vitro culture, implantation acceptor and target gene modify the cultivation of animal.
In step 2, the qualification of described hypertension mouse animal model comprises:
(1) cervical dislocation sacrifices mouse, extracts the liver of F1 generation chimeric mice and wild control mice, skeletal muscle, fat, kidney and spinal cord total serum IgE; Reverse transcription becomes cDNA; Amplifying target genes, compares the STK39 gene expression difference of heterozygote and wild control mice;
(2) CRISPR target site is carried out to the genotype of DNA sequencing qualification mouse model;
(3) real-time quantitative PCR is utilized to detect the change of STK39 gene mRNA levels in mouse cell;
(4) disappearance of STK39 albumen in mouse cell is detected by immunofluorescence dyeing.
the analysis of embodiment 2 Relevant phenotype
1, the measurement of mouse blood pressure
By the mouse that embodiment 1 obtains, raise and within three to eight months, can be used for carrying out blood pressure measurement.What adopt in the present invention is that caudal artery tiretube process surveys mouse blood pressure, and instrument is the non-invasive blood pressure instrument of Shanghai Alcott.Before experimental data gathers, all will carry out the training of five days by a definite date to experimental group and control group mice, concrete grammar is, at every morning ten to ten one point, every mouse is placed in 32 degree of thermostat containers, surveys ten to two ten minutes continuously.Until start image data after mouse blood pressure Measurement sensibility, every mouse surveys five days continuously, averages as the pressure value of this mouse, knocks out group and control group mice is respectively surveyed ten mouse and added up, and concrete outcome as shown in Figure 1.
2, vascular smooth muscle tension detection
The muscular tension of resistance vessel is by the four-way muscular tension instrument record of DMT instrument company of Denmark, concrete grammar is: separated from mesentery reticular tissue by the mesentery secondary blood vessel of mouse, be cut into the vascular strip of 1.4 millimeters long, then penetrate lumen of vessels with the light gage wire of two 20 micron diameters, be screwed and be connected to the organizing in bath of sensor.Then vascular strip is balanced 30 minutes with the state loosened most in HEPES-Tyrode (H-T) physiological solution, progressively intravascular pressure is transferred to the tension state of 100mmHg in body by adjusting screw micrometer, as initial tension.After being transferred to basal tension state, balancing 30 minutes, then use 124mM Repone K H-T solution or norepinephrine, the agonists such as Angiotensin stimulate blood vessel by tension pick-up real time record vasoconstriction power, and result as shown in Figure 2.
3, renin-angiotensin system measures
STK39 knocks out feritin in the mice serum of group and control group, aldosterone, Angiotensin are detected by business-like ELSIA test kit.Detailed process is as follows: first from capillary vessel clump retina take a blood sample to be added with antithrombotics EDTA pipe in, 1000g, 4 degree centrifugal 15 minutes, get supernatant, packing frozen in-80 degree refrigerators.When being ELISA and detecting, serum is added in 96 orifice plates of feritin, aldosterone or Angiotensin, develop the color with horseradish peroxidase method after hatching washing, under 450nm wavelength, absorbance value is read by microplate reader, converse the feritin in each sample, aldosterone or Angiotensin concentration according to typical curve, result as shown in Figure 3.
4, heart function detects
The cardiac functional parameter of mouse is detected by Vevo 770TM animalcule ultrasonic image-forming system.By mouse with after A Foting anesthesia, be placed on 37 degree of warm tables, then detect parasternal short axis view M type ultrasonoscopy with RMV707B probe.Read M type ultrasonoscopy to measure through tangent plane in left ventricle, calculate through parameter in ejection fraction, minor axis shrinking percentage, left ventricle, result as shown in Figure 4.
5, part hypertension drug detects
In order to detect the drug effect of antihypertensive drugs, drug injection is knocked out to STK39 in the Mice Body of group and control group by the method for tail vein injection by we, then Measure blood pressure changing conditions.Medicine through detecting has amlodipine besylate tablets, perindopril tablets.Wherein, in the mouse that amlodipine besylate tablets knocks out at STK39, antihypertensive effect is not obvious, and the blood pressure of the mouse that perindopril tablets can make contrast and knock out group all reduces, and result as shown in Figure 5.
Above specific embodiments of the invention are described.It is to be appreciated that the present invention is not limited to above-mentioned particular implementation, those skilled in the art can make various distortion or amendment within the scope of the claims, and this does not affect flesh and blood of the present invention.
Claims (7)
1. set up a method for hypertension mouse model, be the method setting up hypertension mouse model based on CRISPR/Cas9 gene Knockout, it is characterized in that, described method comprises the steps:
Step one, set up STK39 gene knock-out mice model;
The qualification of step 2, hypertension mouse animal model;
The Relevant phenotype analysis of step 3, hypertension mouse animal model.
2. the method setting up hypertension mouse model according to claim 1, is characterized in that, in step one, describedly sets up STK39 gene knock-out mice model, specifically comprises the following steps:
The structure of A, CRISPR targeting modification genophore;
B, mouse induced ovulation and in vitro fertilization;
The microinjection of C, mouse fertilized egg;
D, zygote vitro culture, implantation acceptor and target gene modify the cultivation of animal.
3. the method setting up hypertension mouse model according to claim 1, is characterized in that, in step 2, the qualification of described hypertension mouse animal model comprises:
(1) cervical dislocation sacrifices mouse, extracts the liver of F1 generation chimeric mice and wild control mice, skeletal muscle, fat, kidney and spinal cord total serum IgE; Reverse transcription becomes cDNA; Amplifying target genes, compares the STK39 gene expression difference of heterozygote and wild control mice;
(2) CRISPR target site is carried out to the genotype of DNA sequencing qualification mouse model;
(3) real-time quantitative PCR is utilized to detect the change of STK39 gene mRNA levels in mouse cell;
(4) disappearance of STK39 albumen in mouse cell is detected by immunofluorescence dyeing.
4. the method setting up hypertension mouse model according to claim 1, is characterized in that, in step 3, the Relevant phenotype analysis of described hypertension mouse animal model comprises:
1) Mouse tail artery pressure is measured;
2) by the heart function of small animal imaging systems axiol-ogy mouse;
3) concentration of feritin, Angiotensin, aldosterone in mice serum is detected by ELISA kit;
4) shrink tension of mouse resistance vessel is measured with muscular tension instrument;
5) tail arterial blood pressure of mouse is detected after tail vein injection hypertension drug.
5. the application of method in high blood pressure disease research of setting up hypertension mouse model according to claim 1.
6. the application of method in the screening for the treatment of hypertension drug of setting up hypertension mouse model according to claim 1.
7. the method setting up hypertension mouse model according to claim 1 is setting up the application in fixed point mutant mouse.
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Cited By (6)
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| CN107475300A (en) * | 2017-09-18 | 2017-12-15 | 上海市同济医院 | The construction method of Ifit3 eKO1 knock out mice animal models and application |
| CN108486154A (en) * | 2018-04-04 | 2018-09-04 | 福州大学 | A kind of construction method of sialidase gene knock-out mice model and its application |
| CN109022485A (en) * | 2018-08-16 | 2018-12-18 | 华东师范大学 | A kind of construction method, kit and the application of optic atrophy animal model |
| CN109777828A (en) * | 2018-12-28 | 2019-05-21 | 江苏集萃药康生物科技有限公司 | A kind of genetic modification animal model preparation method based on IVF Yu CRISPR/Cas9 gene editing technology |
| CN112522312A (en) * | 2020-11-23 | 2021-03-19 | 福建省立医院 | WKH rat model construction method |
| CN114032237A (en) * | 2021-10-11 | 2022-02-11 | 哈尔滨医科大学 | Circular non-coding RNA circSTK39 and application thereof in preventing and treating atherosclerosis |
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Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN107475300A (en) * | 2017-09-18 | 2017-12-15 | 上海市同济医院 | The construction method of Ifit3 eKO1 knock out mice animal models and application |
| CN107475300B (en) * | 2017-09-18 | 2020-04-21 | 上海市同济医院 | Construction method and application of Ifit3-eKO1 gene knockout mouse animal model |
| CN108486154A (en) * | 2018-04-04 | 2018-09-04 | 福州大学 | A kind of construction method of sialidase gene knock-out mice model and its application |
| CN109022485A (en) * | 2018-08-16 | 2018-12-18 | 华东师范大学 | A kind of construction method, kit and the application of optic atrophy animal model |
| CN109022485B (en) * | 2018-08-16 | 2021-02-02 | 华东师范大学 | Construction method, kit and application of optic atrophy animal model |
| CN109777828A (en) * | 2018-12-28 | 2019-05-21 | 江苏集萃药康生物科技有限公司 | A kind of genetic modification animal model preparation method based on IVF Yu CRISPR/Cas9 gene editing technology |
| CN112522312A (en) * | 2020-11-23 | 2021-03-19 | 福建省立医院 | WKH rat model construction method |
| CN112522312B (en) * | 2020-11-23 | 2022-10-04 | 福建省立医院 | WKH rat model construction method |
| CN114032237A (en) * | 2021-10-11 | 2022-02-11 | 哈尔滨医科大学 | Circular non-coding RNA circSTK39 and application thereof in preventing and treating atherosclerosis |
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Application publication date: 20150121 |