CN117568398B - Method for constructing arrhythmia animal model by PGC-1 alpha gene knockout mice and application thereof - Google Patents
Method for constructing arrhythmia animal model by PGC-1 alpha gene knockout mice and application thereof Download PDFInfo
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Abstract
The invention relates to a method for constructing an arrhythmia animal model by a PGC-1 alpha gene knockout mouse and application thereof, wherein the method comprises the following steps: 1) Synthesizing specific target site gRNA of PGC-1 alpha gene: 2) Constructing a targeting vector: 3) Uniformly mixing the reverse strand sequence of the Cas9mRNA and the gRNA, the positive strand sequence of the gRNA and the targeting vector Donor vector to obtain an injection compound, microinjecting the injection compound into fertilized eggs of mice, culturing and passaging to obtain the compound. The mice with conditional PGC-1 alpha knockdown of the invention provide a reliable animal model for the mechanism research of some diseases caused by mitochondrial injury. Conditional knockout of PGC-1 alpha mice can determine the cell type of PGC-1 alpha gene deletion, and more objectively and systematically study the action and mechanism of PGC-1 alpha gene in different tissue organogenesis, development or disease occurrence and treatment processes.
Description
Technical Field
The invention belongs to the technical field of gene editing, and particularly relates to a method for constructing an arrhythmia animal model by using a PGC-1 alpha gene knockout mouse and application thereof.
Background
Arrhythmia refers to an abnormality in the frequency, rhythm, origin, conduction velocity, or activation sequence of heart impulses, and is a clinically extremely common disease, one of the main causes of Sudden Cardiac Death (SCD) (sudden CARDIAC DEATH). The cause of arrhythmia onset is mainly divided into hereditary and acquired. Genetic arrhythmia, mostly ion channel disease caused by gene mutation, causes abnormal ion flow of cardiac muscle cells. Acquired, physiological factors related to motor, emotional changes, and pathological factors caused by heart itself, systemic and other organ disorders. SCD caused by arrhythmia can account for 25% of all deaths, and can be treated by drug therapy, cardioversion therapy or surgery, but the incidence rate of SCD in China is 41.84/10 ten thousand, the number of sudden death per year reaches 54.4 ten thousand, the world is the first place, and the disease burden is serious. The current commonly used models are a slow arrhythmia model, a fast arrhythmia model, an atrial flutter and tremor arrhythmia model, a ventricular tachycardia and ventricular tremor arrhythmia model, an atrioventricular block and atrioventricular junction region conduction normal arrhythmia model and a sinus node arrhythmia model, but the construction of an arrhythmia animal model by using a CRISPR/Cas9 system conditional knockout peroxisome proliferator activated receptor gamma co-activated factor 1 alpha (PGC-1 alpha) gene mouse has not been clinically applied.
The animal model gene editing technology is mainly divided into two categories: ES cell targeting techniques and CRISPR/Cas9 techniques. Wherein the ES cell targeting is to conduct DNA homologous recombination in mouse embryonic stem cells (ES cells), re-inject the ES cells into blastula cavity to form chimeric embryo, and develop chimeric mice in pseudopregnant mice. The chimeric mice were then mated with wild-type mice, thereby transferring genetic information in the ES cells to offspring mice. Its advantages are no off-target effect, high effect, low efficiency, time consumption, and high cost. Thus, the most widely used at present is the CRISPR/Cas9 system. The system is composed of Cas9 protein and gRNA as cores, wherein Cas9 contains RuvC at the tail end of an amino group and HNH at the middle part of the protein, and plays roles in crRNA maturation and double-strand DNA shearing to cause DNA double-strand break. When DNA breaks, DNA fragments homologous to damaged DNA exist in the nucleus at the same time, exogenous DNA fragments can be introduced into target sites through homologous-mediated double-stranded DNA repair (HDR) to achieve the effect of fragment knock-in or editing. Its advantages are high effect, high speed, simple and convenient operation, low cost and application to different species. The disadvantage is that there is always an unpredictable and uncontrollable off-target effect, but with the accuracy of the gRNA design and Cas9 nuclease mutation, the off-target effect has been greatly reduced.
PGC-1 α is a nuclear receptor-assisted activator which has been attracting attention in recent years, and by interacting with nuclear receptors such as PPARs, NRFs and ERRs, it regulates mitochondrial biosynthesis, substance and energy metabolism and antioxidant stress, and plays an important role in mitochondrial biosynthesis, skeletal muscle fiber type conversion, body-adaptive thermogenesis, glycometabolism and lipid metabolism, and is involved in pathophysiological processes of cardiovascular diseases such as cardiac hypertrophy, heart failure, cardiomyopathy, atherosclerosis and myocardial ischemia. The animal model is an important basis for developing new drugs and new therapies, and a good animal model can provide a good platform for further researching pathogenesis of arrhythmia, evaluating the effect of therapeutic means and other clinical researches.
In view of the important role played by PGC-1 alpha in the pathophysiological process of cardiovascular diseases, the invention provides a construction method of a PGC-1 alpha gene knockout mouse animal model and application thereof aiming at the improvement technical scheme of the defects of the prior art.
Disclosure of Invention
Aiming at the defects of the prior art, the invention aims to provide a construction method and application of a PGC-1 alpha gene knockout mouse animal model.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides a method for constructing an arrhythmia animal model by using a PGC-1 alpha gene knockout mouse, which comprises the following steps:
(1) Synthesizing specific target site gRNA of PGC-1 alpha gene:
The gRNA includes an inverted strand sequence and a positive strand sequence; the nucleotide sequence of the reverse chain sequence is shown as SEQ ID NO. 1, and the nucleotide sequence of the positive chain sequence is shown as SEQ ID NO. 2;
(2) Constructing a targeting vector: the nucleotide sequence of the Donor vector is shown as SEQ ID NO. 9;
(3) Microinjection: uniformly mixing the reverse strand sequence of the Cas9 mRNA and the gRNA, the positive strand sequence of the gRNA and the targeting vector Donor vector to obtain an injection compound, microinjecting the injection compound into a mouse fertilized egg, and culturing and passaging the injection compound to obtain the PGC-1 alpha gene knockout mouse animal model.
Preferably, the Gene ID of the PGC-1. Alpha. Gene: 19017.
Preferably, the target site gRNA is located at exons 4-5 of the PGC-1 alpha gene.
Preferably, the specific method for the passage is as follows: 1) Extracting DNA of the tail of the F0 generation mouse, carrying out PCR identification to obtain a positive mouse, and mating the positive mouse with a wild type mice to obtain an F1 generation mouse; 2) Extracting DNA of the tail of the F1 generation mouse, and carrying out PCR identification to obtain a mouse with a genotype of PGC-1 alpha flox+/-, and mating the mouse with a wild type foreign mouse to obtain an F2 generation mouse; 3) Selecting PGC-1 alpha flox+/- mice from F2 mice, and mutually inbreeding the PGC-1 alpha flox+/- mice to generate F3 mice, so as to obtain a stable PGC-1 alpha flox+/+ mouse strain; 4) PGC-1 alpha flox+/+ mice were crossed with different species of cre mice to give conditional knockout mice.
Preferably, the wild-type mice are wild-type C57BL/6 mice;
Preferably, the primer sequences involved in the PCR identification of the F0-generation mice or the F1-generation mice comprise a PCR primer 1 and a PCR primer 2; the nucleotide sequence of the primer upstream of the PCR primer 1 is shown as SEQ ID NO. 11; the nucleotide sequence of the primer downstream of the PCR primer 1 is shown as SEQ ID NO. 12; the nucleotide sequence of the primer upstream of the PCR primer 2 is shown as SEQ ID NO. 13; the nucleotide sequence of the primer downstream of the PCR primer 2 is shown as SEQ ID NO. 14.
Preferably, the primer sequences involved in the PCR identification of the F2-generation mice or the F3-generation mice comprise an upstream primer F4 and a downstream primer R4; the nucleotide sequence of the upstream primer F4 is shown as SEQ ID NO. 21; the nucleotide sequence of the downstream primer R4 is shown as SEQ ID NO. 22.
Preferably, the primer sequences involved in the PCR identification of the conditional knockout mice in the step 4) comprise an upstream primer Myh6-Cre-M-F and a downstream primer Myh6-Cre-M-R; the nucleotide sequence of the upstream primer Myh6-Cre-M-F is shown as SEQ ID NO. 23; the nucleotide sequence of the downstream primer Myh6-Cre-M-R is shown as SEQ ID NO. 24.
The invention provides an application of a PGC-1 alpha gene knockout mouse animal model obtained by the method in researching diseases caused by mitochondrial injury.
The invention provides an application of a PGC-1 alpha gene knockout mouse animal model obtained by the method in preparing antiarrhythmic drugs.
The beneficial effects are that:
The gRNA designed by the invention can obviously improve the specificity of genome editing, and has no off-target effect; the cKO region selects the exons 4-5 of the PGC-1 alpha gene of the mouse model as the cKO region, so that the downstream open reading frame of the transcribed transcript is shifted, and deleting the region can lead to the loss of the function of the PGC-1 alpha gene of the mouse, thereby achieving the experimental effect of gene knockout, and having lower cost, high success rate and high practicability; after the Donor vector, the gRNA and the Cas9mRNA constructed by the invention are microinjected into fertilized eggs, high-efficiency homologous recombination can be carried out with a target site by a homologous recombination mode, so that the DNA large fragment knock-in and conditional PGC-1 alpha knockout can be realized.
The invention establishes the PGC-1 alpha flox+/+ mice containing Loxp sites based on the CRISPR/Cas9 system, and mates with cre mice with different specificities, so that the PGC-1 alpha mice with different tissue specificities or induced knockout of different tissue specificities can be obtained to construct arrhythmia models, thereby saving the cost and providing convenience for researching the specific effect of PGC-1 alpha in diseases.
The mice with conditional PGC-1 alpha knockdown of the invention provide a reliable animal model for the mechanism research of some diseases caused by mitochondrial injury. Conditional knockout of PGC-1 alpha mice can determine the cell type of PGC-1 alpha gene deletion, and more objectively and systematically study the action and mechanism of PGC-1 alpha gene in different tissue organogenesis, development or disease occurrence and treatment processes. Therefore, the PGC-1 alpha conditional gene knockout mouse has wide application prospect, for example, is helpful for the research and development of antiarrhythmic drugs.
Drawings
The accompanying drawings, which are included to provide a further understanding of the application and are incorporated in and constitute a part of this specification, illustrate embodiments of the application and together with the description serve to explain the application. Wherein:
FIG. 1 is a schematic diagram of the procedure for conditional knockout of PGC-1 alpha mice in accordance with the CRISPR/Cas9 technology of the present invention.
FIG. 2 is a graph showing the results of the digestion verification of the targeting vector of the present invention.
FIG. 3 is a schematic diagram showing the design position of a long-chain PCR primer across a5 'arm or a 3' arm and an insert in an F1-generation mouse according to the present invention.
FIG. 4 is a graph showing the identification result of F1-generation mice in which loxP site is inserted into the 5' -end of the present invention.
Wherein 2,3, 4 in fig. 4 represent the F1 generation mouse samples, respectively, M represents DNA MARKER, WT wild type control group samples, water represents Water blank control samples, and fig. 5 is the same.
FIG. 5 is a graph showing the identification result of F1-generation mice in which loxP site is inserted into the 3' -end of the present invention.
FIG. 6 is a diagram showing the sequencing verification of loxP site in F1 mice of the present invention.
FIG. 7 is a schematic diagram of Southern blot analysis of F1 mice of the present invention.
FIG. 8 is a graph showing the result of Southern blot identification of F1 mice of the present invention.
Wherein 2, 3, 4 represent F1 generation mouse samples, respectively, and WT represents a wild type control group sample.
FIG. 9 is a graph showing the results of the identification of PGC-1α (PGC-1α flox+/+,myh6) mice with myocardial specific knockout according to the present invention.
FIG. 10 is a schematic diagram of the propagation of mice according to the present invention.
FIG. 11 is a representation of ECG representations of PGC-1α flox+/+ mice and PGC-1α flox+/+,myh6 mice of the present invention.
Wherein, control in the figure represents PGC-1 alpha flox+/+ mice; model represents PGC-1 alpha flox+/+,myh6 mice.
FIG. 12 is a graph representing ventricular conductance of PGC-1α flox+/+ mice and PGC-1α flox+/+,myh6 mice of the present invention.
FIG. 13 is a graph showing the field potential representation of PGC-1α flox+/+ mice and PGC-1α flox+/+,myh6 mice according to the present invention.
FIG. 14 is an arrhythmia induction graph of PGC-1α flox+/+ mice and PGC-1α flox+/+,myh6 mice of the present invention.
Detailed Description
The following description of the technical solutions in the embodiments of the present invention will be clear and complete, and it is obvious that the described embodiments are only some embodiments of the present invention, but not all embodiments. All other embodiments, which are derived by a person skilled in the art based on the embodiments of the invention, fall within the scope of protection of the invention.
The present invention will be described in detail with reference to examples. It should be noted that, without conflict, the embodiments of the present invention and features of the embodiments may be combined with each other.
Aiming at the existing problems, the invention provides a method for constructing an arrhythmia animal model by using a PGC-1 alpha gene knockout mouse, which comprises the following steps:
(1) Synthesizing specific target site gRNA of PGC-1 alpha gene:
The gRNA includes an inverted strand sequence and a positive strand sequence; the nucleotide sequence of the reverse chain sequence is shown as SEQ ID NO. 1, and the nucleotide sequence of the positive chain sequence is shown as SEQ ID NO. 2;
(2) Constructing a targeting vector: the nucleotide sequence of the Donor vector is shown as SEQ ID NO. 9;
(3) Microinjection: uniformly mixing the reverse strand sequence of the Cas9 mRNA and the gRNA, the positive strand sequence of the gRNA and the targeting vector Donor vector to obtain an injection compound, microinjecting the injection compound into a mouse fertilized egg, and culturing and passaging the injection compound to obtain the PGC-1 alpha gene knockout mouse animal model.
In a preferred embodiment of the present invention, the Gene ID of the PGC-1. Alpha. Gene: 19017; the target site gRNA is located at the 4 th-5 th exons of PGC-1 alpha gene.
In a preferred embodiment of the invention, the specific method for the passage is as follows:
1) Extracting DNA of the tail of the F0 generation mouse, carrying out PCR identification to obtain a positive mouse, and mating the positive mouse with a wild type mice to obtain an F1 generation mouse; 2) Extracting DNA of the tail of the F1 generation mouse, and carrying out PCR identification to obtain a mouse with a genotype of PGC-1 alpha flox+/-, and mating the mouse with a wild type foreign mouse to obtain an F2 generation mouse; 3) Selecting PGC-1 alpha flox+/- mice from F2 mice, and mutually inbreeding the PGC-1 alpha flox+/- mice to generate F3 mice, so as to obtain a stable PGC-1 alpha flox+/+ mouse strain; 4) PGC-1 alpha flox+/+ mice were crossed with different species of cre mice to give conditional knockout mice.
In a preferred embodiment of the invention, the wild-type mice are wild-type C57BL/6 mice;
In a preferred embodiment of the invention, the primer sequences involved in the PCR identification of the F0-generation mice or the F1-generation mice comprise a PCR primer 1 and a PCR primer 2; the nucleotide sequence of the primer upstream of the PCR primer 1 is shown as SEQ ID NO. 11; the nucleotide sequence of the primer downstream of the PCR primer 1 is shown as SEQ ID NO. 12; the nucleotide sequence of the primer upstream of the PCR primer 2 is shown as SEQ ID NO. 13; the nucleotide sequence of the primer downstream of the PCR primer 2 is shown as SEQ ID NO. 14.
In a preferred embodiment of the invention, the primer sequences involved in the PCR identification of the F2-generation mice or the F3-generation mice comprise an upstream primer F4 and a downstream primer R4; the nucleotide sequence of the upstream primer F4 is shown as SEQ ID NO. 21; the nucleotide sequence of the downstream primer R4 is shown as SEQ ID NO. 22.
In a preferred embodiment of the invention, the primer sequences involved in the PCR identification of the conditional knockout mice in the step 4) comprise an upstream primer Myh6-Cre-M-F and a downstream primer Myh6-Cre-M-R; the nucleotide sequence of the upstream primer Myh6-Cre-M-F is shown as SEQ ID NO. 23; the nucleotide sequence of the downstream primer Myh6-Cre-M-R is shown as SEQ ID NO. 24.
The invention provides an application of a PGC-1 alpha gene knockout mouse animal model obtained by the method in researching diseases caused by mitochondrial injury.
The invention provides an application of a PGC-1 alpha gene knockout mouse animal model obtained by the method in preparing antiarrhythmic drugs.
The method for constructing an animal model of arrhythmia by using the PGC-1 alpha gene knockout mouse and the application thereof are described in detail below by way of specific examples.
Example 1 construction of PGC-1 alpha Gene conditional knockout mice Using CRISPR/Cas9 technology
1. Design of gRNA target sequences
PGC-1 alpha gene (NCBI Reference Sequence: NM-008904;
Ensembl: ENSMUSG 00000029167) is located on mouse chromosome 5. Thus, exons 4-5 were selected as conditional knockdown regions (cKO region). The deletion of this region results in the deletion of the mouse Ppargc a (PGC-1α) gene function.
The specific target sites gRNA of the Gene PGC-1α to be knocked out (Gene ID: 19017) were determined, the DNA sequence of the mouse PGC-1α Gene was found in the mouse genome database (TRANSCRIPT ID: ENSMUST 00000132734.7), and then using on-line design software CRISPOR (http:// crispor. Tefor. Net), it was determined to select 2 specific sites within target sites exon 4 and 5 of the mouse PGC-1α Gene as target sequences of gRNA, respectively: REVERSE STRAND (inverted strand) sequence: GCTGGCCCACCAATGCTTTGAGG (SEQ ID NO: 1), forward strand (plus strand) sequence: CAAGGCACATTCGGTGATTTGGG (SEQ ID NO: 2).
CRISPR/cas mediated genome engineering an overall protocol for creating PGC-1 a conditional knockout mice models is shown in figure 1.
2. Construction of a Donor vector
(1) A mouse genome fragment containing the Homology arm (Homology arm) and conditional knockdown (cKO) region was amplified from BAC: RP23-358P6 clone template using high fidelity Taq DNA polymerase (Norvirzan P515) as follows:
Cloning a template: 50ng; primer F/R: 3uL each; sterilizing water: 40uL; 2X Phanta Max Master Mix (P515) 50uL; after mixing and sub-packaging the mixture for two tubes, the PCR reaction was performed in a total volume of 100 uL. The reaction conditions were as follows: 95 ℃ for 5min.95℃for 10s,60℃for 15s,72℃for 1kb/min (30 cycles). Storing at 72deg.C for 10min and 4deg.C.
Wherein the primer sequences are as follows:
5'arm(981bp)F：tatagggcgaattgggtacggcgcgcctgaaatctgtggtaggcaatgg(SEQ ID NO:3)；
5'arm R：
TCGAACTTGTGGCCGTTTACGTACACGTGAGCTCTTTGAGGGGCAGAGGTTAGGAA(SEQ ID NO:4)：
cKO(2497bp)F：
gtaaacggccacaagttcgaataacttcgtatagcatacattatacgaagttatcattggtgggccagccagcctgatattgc(SEQ ID NO:5)；
cKO R：
CGGTTACCGTGGATTCGGACCAGTCTGACATAACTTCGTATAATGTATGCTATACGAAGTTATCACCGAATGTGCCTTGGAAGC(SEQ ID NO:6)；
3'arm(1063bp)F：
tccgaatccacggtaaccgatatcatttggggcaaatctctcttggcagagaaacactttttctctctcacctcttcctattcc(SEQ ID NO:7)；
3'arm R：TGGGCCCTGGTACCAGAATGCGGCCGCATCAGGAGGAGCCTTGGACACTT(SEQ ID NO:8)；
(2) The mouse genome fragment, loxp (locus of X-over P1) was assembled together with the modified PUC57 backbone (supplied by Seikovia Biotechnology Co., ltd.) to form a Donor vector. And (3) converting the assembled Donor vector into competent cells, inoculating bacteria positively by a bacterial screen, extracting plasmids, carrying out enzyme digestion identification, and carrying out sequencing. The assembly was done by the company of the Celite Biotechnology Co., ltd, the method was as follows: modified PUC57 backbone 100ng, mouse genome ligation fragment 50ng, 2X ClonExpress Mix. Mu.L (Northene, C115), and sterile water to 10. Mu.L. 50℃for 15min.
The Donor vector sequence (SEQ ID NO: 9) is shown below, with the underlined as the Homology arm, the double underlined as the cKO region, the wavy line as the loxP site, and the black bolded as the Exon.
The Donor vector sequence:
(3) In order to verify the correctness of the whole vector, the digestion verifies the targeting vector, the size and the position of the bands are correct after the electrophoresis detection is carried out by using different enzymes, and the result is shown in figure 2. Wherein, fspI (NEB, R0135V) enzyme is used for enzyme digestion with the size of 3.4/2.4/1.7/1.2kb; cleavage was performed with an AflII (NEB, R0520V)/AvaI (NEB, R0152V) enzyme at a size of 4.2/2.8/0.9/0.6/0.1kb; the enzyme digestion was carried out with AhdI (NEB, R0584V)/DrdI (NEB, R0530V) enzyme at a size of 5.3/1.5/1.1/0.8kb; the cleavage was performed with NotI (NEB, R0189V) enzyme at a size of 8.7kb.
3. Microinjection
Cas9 mRNA, gRNA and a Donor vector (containing loxP sites) were mixed uniformly using microinjection technique, and injected into the prokaryote of fertilized egg, and then fertilized egg was transferred into pseudopregnant mother oviduct. The fertilized eggs continued to develop into individuals, and rat tail identification was performed 7 days after birth of the mice, so as to obtain positive F0 generation Founder mice (identification method is the same as the following 4, identification and screening of PGC-1 alpha flox+/- mice). The Cas9 protein cuts the DNA double chain of the PGC-1 alpha gene under the targeting action of gRNA, so that a homologous recombination mechanism is induced to repair the damaged PGC-1 alpha gene by taking the Donor DNA as a template, and LoxP sequences are integrated into specific sites in the genome of the mouse.
Preparation of Cas9 mRNA, gRNA and dosator injection complexes: comprising a tube 1 solution and a tube 2 solution, and mixing the tube 1 solution and the tube 2 solution to obtain the RNP injection complex.
Tube 1 solution: adding CrRNA to 0.8uL 100pmol/uL of RNase-free water (RNase-free water), adding TracRNA to 0.6uL 100pmol/uL, mixing, incubating for 5min, and adding 0.2uL of Cas9 proteinCas9 NLS, s.pyogens, NEB, cat: M0646M) and incubating for 10min to obtain a tube 1 solution;
tube 2 solution: donor vector plasmid with final concentration of 15 ng/uL;
wherein, tracRNA (INTEGRATED DNA Technologies, IDT), sequence:
5'-AAACAGCAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCU-3' (SEQ ID NO:10, if represented by ST.26, uracil "U" in the sequence is represented by "t"); crRNA (synthesized by Nanjing Jinsri Biotechnology Co., ltd.) comprises a gRNA reverse strand sequence (SEQ ID NO: 1) and a forward strand sequence (SEQ ID NO: 2).
4. Identification and screening of mice with genotype PGC-1 alpha flox+/-
Because of the unstable nature of F0 generation, C57BL/6 females are required to obtain stably inherited F1 mice. After the F0 generation mice are born, selecting mice with loxP inserted into the 5 'end and the 3' end, and mating the mice with wild C57BL/6 female mice after the mice are grown to obtain the F1 generation mice. The positive expression of the loxP-cKO region-homology arm was verified by PCR. FIG. 3 is a schematic diagram of the design position of a long-chain PCR primer across the 5 'arm or 3' arm and insert in F1 mice.
(1) Extraction of mouse tissue genomic DNA:
① The mice were marked with ear tags, the tail was cut to 2-5mm tissue, and placed in a 1.5mL pointed Eppendorf (Ai Bende) centrifuge tube. ② To a 1.5mL Eppendorf centrifuge tube containing tissue was added 100. Mu.L of buffer with final concentration of 50mM KCl, 10mM Tris-HCl, 0.1% Triton X-100, 0.4mg/mL Proteinase K (Proteinase K) and incubated overnight at 56 ℃. ③ The tube was incubated at 98℃for 13 min to denature proteinase K. ④ The microcentrifuge was spun at high speed for 15 minutes. Aliquots of the supernatant (2. Mu.L in 50. Mu.L reaction) were taken directly from the tube for PCR.
(2) And (3) PCR amplification:
① PCR reaction
| Component (A) | Addition of |
| Mouse tail genomic DNA | 2μL |
| Forward primer(10μM) | 2μL |
| Reverse primer(10μM) | 2μL |
| dNTPs(2.5mM) | 6μL |
| 5X LongAmp Taq Reaction | 10μL |
| LongAmp Taq DNA Polymerase | 2μL |
| ddH2O | 26μL |
| Totals to | 50μL |
② Amplification conditions: 94℃for 3min, (94℃for 30s,60℃for 30s,65℃for 50 s). Times.33 cycles,65℃for 10min.
③ Primer sequence:
PCR primer 1:
5’arm forward primer(F1):5’-AACAATAATAGCTCTTCTGCAGCC-3’(SEQ ID NO:11)；
3’loxP reverse primer(R1):5’-CCAAATGATATCGGTTACCGTGGA-3’(SEQ ID NO:12)。
PCR primer 2:
5’loxP forward primer(F2):5’-ACGTAAACGGCCACAAGTTC-3’(SEQ ID NO:13)；
3’arm reverse primer(R2):5’-AGAGCCTAAAACTCTCAGTTCAGC-3’(SEQ ID NO:14)。
Sequencing and verifying primers:
5’loxP Sequence primer(F3):5’-ACCACACCAATGAAGAAACTGGG-3’(SEQ ID NO:15)；
3’loxP Sequence primer(R3):5’-TAGAGAACCGGAAACACACGAG-3’(SEQ ID NO:16)。
5' primer probe:
5’Probe forward primer：5’-CTGAAGGAACTTGACATGGGCAAA-3’(SEQ ID NO:17)；
5’Probe reverse primer：5’-TGCCTGGATGATAGGTATGCGTTACA-3’(SEQ ID NO:18)；
3' primer probe:
3’Probe forward primer：5’-GGAGGGATCAACTGAAAGAAGGTACG-3’(SEQ ID NO:19)；
3’Probe reverse primer：5’-CAAGCACACTTACTGGGTGGGAAAT-3’(SEQ ID NO:20)。
(3) Identification result of F1 generation PGC-1 alpha flox+/-
The genomic DNA of F1-generation mouse tissues was verified by using PCR primer 1 and PCR primer 2, respectively, and the results are shown in FIGS. 4 and 5. The positive sample of the F1/R1 primer amplification product in FIG. 4 was 3.5kb, and the positive sample of the F2/R2 primer amplification product in FIG. 5 was 3.6kb. The size and the position of the PCR product fragments of 3F 1 mice are correct, and the genotypes of the 3F 1 mice can be judged to be PGC-1 alpha flox+/-.
(4) Sequencing to verify that the loxp sequence is correct
The F1 mice were subjected to sequencing verification (sequencing verification primer F3/R3), and the results are shown in FIG. 6. By aligning the sequencing sequences of loxP sites, the loxP sites are identical to the target fragment.
(5) Southern blot (blotting hybridization technique) identification
Southern blot identification was performed on F1 mice, the Southern blot analysis schematic diagram is shown in FIG. 7, and the Southern blot identification result is shown in FIG. 8. As can be seen from FIG. 8, the 5' -end probe PCR amplified after SacI cleavage was designed to give probe markers: the wild-type mouse fragment size was 7.16kb, the PGC-1α flox+/- fragment size was 7.16kb and the 5.41kb, and the mouse PGC-1α flox+/+ mouse fragment size was 5.41kb. And (3) probe labeling amplified by PCR (polymerase chain reaction) of a 3' -end probe designed after BstEII enzyme digestion: the wild-type mouse fragment size was 13.73kb, the PGC-1α flox+/- fragment size was 13.73kb and 5.84kb, and the PGC-1α flox+/+ mouse fragment size was 5.84kb.
F1 generation mice 2, 3, 4 were PGC-1α flox+/- mice, so that the Southern blot resulted in a 5'probe-SacI fragment size of 7.16kb and 5.41kb, and a 3' probe-BstEII fragment size of 13.73kb and 5.84 kb.
5. Propagation and Gene identification
(1) F3 generation PGC-1 alpha flox+/+ mouse identification:
the F1 generation PGC-1 alpha flox+/- mouse is verified to be free of problems, and then the primer can be designed aiming at the loxp locus to identify the PGC-1 alpha flox+/+ mouse. The F1 generation loxp heterozygote mice are hybridized with wild type C57BL/6 mice (namely B6 wild type mice in FIG. 10) to obtain F2 generation mice, PGC-1 alpha flox+/- mice are selected from F2 generation mice, and PGC-1 alpha flox+/- mice are mutually inbred to generate F3 generation mice, so that a stable PGC-1 alpha flox+/+ mouse strain is obtained.
PCR identification was performed using the above method, and the primer design was as follows (both F2 generation and F3 generation are applicable):
F4:5’-CTGATTTCCTCTGCCTTGTGTATTTAG-3’(SEQ ID NO:21)；
R4:5’-TTGGAATAGGAAGAGGTGAGAGAGA-3’(SEQ ID NO:22)。
(2) PGC-1 alpha flox+/+ mice were crossed with different kinds of cre mice to obtain conditional knockout mice, and the propagation diagram is shown in FIG. 10. PCR identification was performed using the above method, and the primers were designed as follows:
Myh6-Cre-M-F:5’-TCTATTGCACACAGCAATCCA-3’(SEQ ID NO:23)；
Myh6-Cre-M-R:5’-CCAGCATTGTGAGAACAAGG-3’(SEQ ID NO:24)。
The results of the identification are shown in FIG. 9, in which wild-type mice (WT mice) have a flox fragment size of 182bp and no myh expression. The flox fragment size in PGC-1 alpha flox+/+ is 250bp, and myh is not expressed. The size of the flox fragment in PGC-1 alpha flox+/+,myh6 mice was 250bp and the size of the myh6 fragment was 300bp, indicating that it expressed myh.
Wherein, PGC-1 alpha flox+/+ mice can be hybridized with Myh6-cre mice to obtain PGC-1 alpha mice with myocardial cell specific knockout. PGC-1 alpha flox+/+ mice can be hybridized with Cdh16-cre mice to obtain kidney-specific knockout PGC-1 alpha mice.
Application example 1 detection of electrophysiological Change in Normal mice and myocardial cell-specific knockout PGC-1 alpha Gene knockout mice
PGC-1α flox+/+,myh6 mice were obtained by hybridization of PGC-1α flox+/+ mice with Myh6-cre (aMHC-MERCREMER) mice. Tamoxifen (40 mg/kg) was continuously injected for 5 days into 8-10 week old male PGC-1α flox+/+ mice (Control group) and PGC-1α flox+/+,myh6 mice (Model group). After the last administration for 4 weeks, the isolated heart of the mouse is hung on a Langendroff perfusion system, residual blood in the heart is discharged, the heart is recovered to normal rhythm, and the experiment is carried out after the heart is stabilized for 15 min; mapping a left ventricle and a right ventricle by adopting 128-channel electrical mapping, and recording field potential signals under sinus and 8HZ stimulation conditions; II leads record Electrocardiogram (ECG), S1S2 stimulation given 2 times threshold current finds effective refractory period, S1S1 stimulation given 2/5/10 times threshold current, and induction of ventricular tachycardia and ventricular fibrillation is counted.
(1) The ECG representation is shown in fig. 11, where panels a, B: ECG representative plots for control and knock-out groups; c, drawing: a heart rate statistics plot; d, drawing: QRS (magnetic resonance angiography) statistics; e, drawing: QT interval (time from QRS complex start to T wave end); f, drawing: ERP (subendocardial resuscitation ratio) statistics; graph G: ERP/QT statistical diagram; drawing H: CV (conduction velocity) ERP statistical plot. Experimental results show that PGC-1 gene knockout mice reduce heart rate, prolong QT and reduce CV.
(2) Ventricular conduction representative diagrams are shown in fig. 12, wherein a diagram a: a cardiac mapping schematic; b, drawing: left ventricle conduction and conduction dispersion representative graph of mice under 8Hz serial stimulation; c, drawing: a left ventricle conduction time statistical graph of the mice under 8Hz serial stimulation; d, drawing: a statistical graph of left ventricular conduction velocity in mice stimulated by 8Hz series; e, drawing: a left ventricle conduction discrete statistical graph of the mice under 8Hz serial stimulation; f, drawing: a statistical graph of right ventricular conduction time of the mice under 8Hz serial stimulation; graph G: a statistical graph of right ventricular conduction velocity of the mice under 8Hz serial stimulation; drawing H: 8Hz series of stimulation with the right ventricle conductive discrete statistical graph. Experimental results show that the PGC-1 gene knockout mouse group increases the dispersion of the left ventricle; reducing left ventricular conduction velocity and right ventricular conduction time; the right ventricle conduction velocity is accelerated, but the method has no statistical significance.
(3) The field potential representation is shown in fig. 13, where a is: a right ventricular field potential statistical map; b, drawing: left ventricular field potential statistical plot. Experimental results show that the PGC-1 gene knockout mouse group prolongs the field potential time course of the left ventricle and the right ventricle.
(4) Arrhythmia induction is shown in fig. 14, where a is: control and model groups S1 induced VT (ventricular rate)/VF (ventricular fibrillation); b, drawing: ECG representative map; c, drawing: a sinoatrial node recovery time statistical graph; d, drawing: a VT/VF total occurrence time statistical diagram; e, drawing: VT/VF occurrence statistics graph: f, drawing: statistical graphs of conduction block occurrence. Experimental results show that the PGC-1 gene knockout mice prolong the sinus node recovery time and increase the occurrence rate of sinus conduction block and ventricular fibrillation chamber speed.
In summary, compared with normal mice, the PGC-1 gene knockout mice reduce heart rate, ERP/QT and CV, prolong the field potential of QT and left/right ventricles, prolong the time of ventricular fibrillation and increase the incidence rate, which indicates that the susceptibility of the PGC-1 gene knockout mice to arrhythmia is increased. The conduction dispersion of the left ventricle increases, the conduction speed is reduced, the conduction dispersion of the right ventricle is unchanged, the conduction speed is increased, and the PGC-1 gene knockout mouse is proved to reduce the function of the left ventricle and compensate the function of the right ventricle. Compared with a normal group, the sinus conduction block of the mice in the gene knockout group is easier to occur in the experimental process, the sinus node recovery time is prolonged when S1S1 is stimulated, which indicates that PGC-1 gene knockout has an effect on the sinus node and the atrioventricular node functions, and the PGC-1 gene knockout mice can reduce heart rate, ERP/QT and CV.ERP, prolong the field potentials of QT and left/right ventricles, and lead to the time extension and the increase of the incidence rate of ventricular fibrillation. These results indicate that PGC-1 alpha down-regulation is a susceptibility gene that induces arrhythmia.
The above description is only of the preferred embodiments of the present invention and is not intended to limit the present invention, but various modifications and variations can be made to the present invention by those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (10)
1. A method for constructing an animal model of arrhythmia by using a PGC-1 alpha gene knockout mouse, comprising the steps of:
(1) Synthesizing specific target site gRNA of PGC-1 alpha gene:
The gRNA includes an inverted strand sequence and a positive strand sequence; the nucleotide sequence of the reverse chain sequence is shown as SEQ ID NO. 1, and the nucleotide sequence of the positive chain sequence is shown as SEQ ID NO. 2;
(2) Constructing a targeting vector:
the nucleotide sequence of the Donor vector is shown as SEQ ID NO. 9;
(3) Microinjection:
Uniformly mixing the reverse strand sequence of the Cas9 mRNA and the gRNA, the positive strand sequence of the gRNA and the targeting vector Donor vector to obtain an injection compound, microinjecting the injection compound into a mouse fertilized egg, and culturing and passaging the injection compound to obtain the PGC-1 alpha gene knockout mouse animal model.
2. The method of claim 1, wherein the PGC-1 a Gene has a Gene ID:19017.
3. The method of claim 1, wherein the target site gRNA targets exons 4-5 of the PGC-1 a gene.
4. The method according to claim 1, wherein the specific method of passaging is:
1) Extracting DNA of the tail of the F0 generation mouse, carrying out PCR identification to obtain a positive mouse, and mating the positive mouse with a wild type mice to obtain an F1 generation mouse;
2) Extracting DNA of the tail of the F1 generation mouse, and carrying out PCR identification to obtain a mouse with a genotype of PGC-1 alpha flox+/-, and mating the mouse with a wild type foreign mouse to obtain an F2 generation mouse;
3) Selecting PGC-1 alpha flox+/- mice from F2 mice, and mutually inbreeding the PGC-1 alpha flox+/- mice to generate F3 mice, so as to obtain a stable PGC-1 alpha flox+/+ mouse strain;
4) PGC-1 alpha flox+/+ mice were crossed with different species of cre mice to give conditional knockout mice.
5. The method of claim 4, wherein the wild-type mouse is a wild-type C57BL/6 mouse.
6. The method of claim 4, wherein the primer sequences involved in the PCR identification of the F0 generation mice or the F1 generation mice comprise PCR primer 1 and PCR primer 2;
the nucleotide sequence of the primer upstream of the PCR primer 1 is shown as SEQ ID NO. 11; the nucleotide sequence of the primer downstream of the PCR primer 1 is shown as SEQ ID NO. 12;
The nucleotide sequence of the primer upstream of the PCR primer 2 is shown as SEQ ID NO. 13; the nucleotide sequence of the primer downstream of the PCR primer 2 is shown as SEQ ID NO. 14.
7. The method of claim 4, wherein the primer sequences involved in the PCR identification of the F2-or F3-generation mice comprise an upstream primer F4 and a downstream primer R4;
the nucleotide sequence of the upstream primer F4 is shown as SEQ ID NO. 21; the nucleotide sequence of the downstream primer R4 is shown as SEQ ID NO. 22.
8. The method of claim 4, wherein the primer sequences involved in step 4) conditional knockout murine PCR identification include an upstream primer Myh6-Cre-M-F and a downstream primer Myh6-Cre-M-R;
the nucleotide sequence of the upstream primer Myh6-Cre-M-F is shown as SEQ ID NO. 23; the nucleotide sequence of the downstream primer Myh6-Cre-M-R is shown as SEQ ID NO. 24.
9. Use of a PGC-1 a knockout mouse animal model obtained by the method of any one of claims 1-8 for studying diseases caused by mitochondrial damage, said use not involving diagnostic and therapeutic methods of disease.
10. Use of a PGC-1 alpha knockout mouse animal model obtained by the method of any one of claims 1-8 in the development of an antiarrhythmic drug, said use not involving diagnostic and therapeutic methods of disease.
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|---|---|---|---|
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