CN117568398A - 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
- Publication number
- CN117568398A CN117568398A CN202311205731.5A CN202311205731A CN117568398A CN 117568398 A CN117568398 A CN 117568398A CN 202311205731 A CN202311205731 A CN 202311205731A CN 117568398 A CN117568398 A CN 117568398A
- Authority
- CN
- China
- Prior art keywords
- pgc
- mice
- primer
- alpha
- seq
- 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.)
- Granted
Links
- 241000699670 Mus sp. Species 0.000 title claims abstract description 132
- 102000017946 PGC-1 Human genes 0.000 title claims abstract description 111
- 108700038399 PGC-1 Proteins 0.000 title claims abstract description 111
- 238000000034 method Methods 0.000 title claims abstract description 38
- 101150087698 alpha gene Proteins 0.000 title claims abstract description 34
- 238000003209 gene knockout Methods 0.000 title claims abstract description 30
- 238000010171 animal model Methods 0.000 title claims abstract description 25
- 206010003119 arrhythmia Diseases 0.000 title claims abstract description 25
- 230000006793 arrhythmia Effects 0.000 title claims abstract description 25
- 241000699666 Mus <mouse, genus> Species 0.000 claims abstract description 58
- 108020005004 Guide RNA Proteins 0.000 claims abstract description 29
- 238000002347 injection Methods 0.000 claims abstract description 13
- 239000007924 injection Substances 0.000 claims abstract description 13
- 150000001875 compounds Chemical class 0.000 claims abstract description 12
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 claims abstract description 12
- 230000008685 targeting Effects 0.000 claims abstract description 12
- 201000010099 disease Diseases 0.000 claims abstract description 11
- 238000002156 mixing Methods 0.000 claims abstract description 7
- 238000012258 culturing Methods 0.000 claims abstract description 4
- 230000002194 synthesizing effect Effects 0.000 claims abstract description 4
- 239000002773 nucleotide Substances 0.000 claims description 33
- 125000003729 nucleotide group Chemical group 0.000 claims description 33
- 108020004414 DNA Proteins 0.000 claims description 20
- 238000011144 upstream manufacturing Methods 0.000 claims description 18
- 108090000623 proteins and genes Proteins 0.000 claims description 16
- 238000011813 knockout mouse model Methods 0.000 claims description 10
- 230000013011 mating Effects 0.000 claims description 8
- 101001123331 Homo sapiens Peroxisome proliferator-activated receptor gamma coactivator 1-alpha Proteins 0.000 claims description 7
- 102100028960 Peroxisome proliferator-activated receptor gamma coactivator 1-alpha Human genes 0.000 claims description 6
- 238000000520 microinjection Methods 0.000 claims description 5
- 108700024394 Exon Proteins 0.000 claims description 4
- 239000003416 antiarrhythmic agent Substances 0.000 claims description 4
- 238000011816 wild-type C57Bl6 mouse Methods 0.000 claims description 4
- 241001529936 Murinae Species 0.000 claims 1
- 230000003288 anthiarrhythmic effect Effects 0.000 claims 1
- 239000003814 drug Substances 0.000 claims 1
- 238000004519 manufacturing process Methods 0.000 claims 1
- 230000006676 mitochondrial damage Effects 0.000 claims 1
- 235000013601 eggs Nutrition 0.000 abstract description 7
- 230000002438 mitochondrial effect Effects 0.000 abstract description 6
- 230000007246 mechanism Effects 0.000 abstract description 5
- 208000027418 Wounds and injury Diseases 0.000 abstract description 4
- 230000006378 damage Effects 0.000 abstract description 4
- 208000014674 injury Diseases 0.000 abstract description 4
- 230000009471 action Effects 0.000 abstract description 3
- 230000008569 process Effects 0.000 abstract description 3
- 238000011160 research Methods 0.000 abstract description 3
- 238000011161 development Methods 0.000 abstract description 2
- 230000018109 developmental process Effects 0.000 abstract description 2
- 238000012224 gene deletion Methods 0.000 abstract description 2
- 230000005305 organ development Effects 0.000 abstract description 2
- 238000003752 polymerase chain reaction Methods 0.000 description 38
- 239000012634 fragment Substances 0.000 description 23
- 108091033409 CRISPR Proteins 0.000 description 17
- 239000000523 sample Substances 0.000 description 13
- 238000010586 diagram Methods 0.000 description 12
- 230000002861 ventricular Effects 0.000 description 11
- 210000005240 left ventricle Anatomy 0.000 description 10
- 210000004027 cell Anatomy 0.000 description 9
- 239000000243 solution Substances 0.000 description 9
- 230000000638 stimulation Effects 0.000 description 9
- 208000003663 ventricular fibrillation Diseases 0.000 description 8
- 238000002105 Southern blotting Methods 0.000 description 7
- 230000000694 effects Effects 0.000 description 7
- 210000005241 right ventricle Anatomy 0.000 description 7
- 238000012163 sequencing technique Methods 0.000 description 7
- 238000010354 CRISPR gene editing Methods 0.000 description 6
- 238000006243 chemical reaction Methods 0.000 description 6
- 210000001519 tissue Anatomy 0.000 description 6
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 6
- 108090000790 Enzymes Proteins 0.000 description 5
- 102000004190 Enzymes Human genes 0.000 description 5
- 238000010276 construction Methods 0.000 description 5
- 238000013461 design Methods 0.000 description 5
- 230000006870 function Effects 0.000 description 5
- 210000001013 sinoatrial node Anatomy 0.000 description 5
- 239000006185 dispersion Substances 0.000 description 4
- 238000005516 engineering process Methods 0.000 description 4
- 238000001976 enzyme digestion Methods 0.000 description 4
- 238000010362 genome editing Methods 0.000 description 4
- 230000006801 homologous recombination Effects 0.000 description 4
- 238000002744 homologous recombination Methods 0.000 description 4
- 239000000203 mixture Substances 0.000 description 4
- 230000009437 off-target effect Effects 0.000 description 4
- 238000012795 verification Methods 0.000 description 4
- 108010067770 Endopeptidase K Proteins 0.000 description 3
- 101100409189 Mus musculus Ppargc1a gene Proteins 0.000 description 3
- 230000003321 amplification Effects 0.000 description 3
- 238000003776 cleavage reaction Methods 0.000 description 3
- 230000006698 induction Effects 0.000 description 3
- 230000002107 myocardial effect Effects 0.000 description 3
- 238000003199 nucleic acid amplification method Methods 0.000 description 3
- 239000000047 product Substances 0.000 description 3
- 238000011084 recovery Methods 0.000 description 3
- 230000007017 scission Effects 0.000 description 3
- FWMNVWWHGCHHJJ-SKKKGAJSSA-N 4-amino-1-[(2r)-6-amino-2-[[(2r)-2-[[(2r)-2-[[(2r)-2-amino-3-phenylpropanoyl]amino]-3-phenylpropanoyl]amino]-4-methylpentanoyl]amino]hexanoyl]piperidine-4-carboxylic acid Chemical compound C([C@H](C(=O)N[C@H](CC(C)C)C(=O)N[C@H](CCCCN)C(=O)N1CCC(N)(CC1)C(O)=O)NC(=O)[C@H](N)CC=1C=CC=CC=1)C1=CC=CC=C1 FWMNVWWHGCHHJJ-SKKKGAJSSA-N 0.000 description 2
- 241000894006 Bacteria Species 0.000 description 2
- 238000011740 C57BL/6 mouse Methods 0.000 description 2
- 208000024172 Cardiovascular disease Diseases 0.000 description 2
- 108020005497 Nuclear hormone receptor Proteins 0.000 description 2
- 206010049418 Sudden Cardiac Death Diseases 0.000 description 2
- NKANXQFJJICGDU-QPLCGJKRSA-N Tamoxifen Chemical compound C=1C=CC=CC=1C(/CC)=C(C=1C=CC(OCCN(C)C)=CC=1)/C1=CC=CC=C1 NKANXQFJJICGDU-QPLCGJKRSA-N 0.000 description 2
- 108010006785 Taq Polymerase Proteins 0.000 description 2
- 206010044565 Tremor Diseases 0.000 description 2
- ISAKRJDGNUQOIC-UHFFFAOYSA-N Uracil Chemical compound O=C1C=CNC(=O)N1 ISAKRJDGNUQOIC-UHFFFAOYSA-N 0.000 description 2
- 230000008901 benefit Effects 0.000 description 2
- 230000015572 biosynthetic process Effects 0.000 description 2
- 239000013068 control sample Substances 0.000 description 2
- 230000007547 defect Effects 0.000 description 2
- 238000012217 deletion Methods 0.000 description 2
- 230000037430 deletion Effects 0.000 description 2
- 238000001514 detection method Methods 0.000 description 2
- 230000029087 digestion Effects 0.000 description 2
- 230000002068 genetic effect Effects 0.000 description 2
- 238000009396 hybridization Methods 0.000 description 2
- 230000006872 improvement Effects 0.000 description 2
- 238000013507 mapping Methods 0.000 description 2
- 230000001404 mediated effect Effects 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 102000006255 nuclear receptors Human genes 0.000 description 2
- 108020004017 nuclear receptors Proteins 0.000 description 2
- 230000035778 pathophysiological process Effects 0.000 description 2
- 239000013612 plasmid Substances 0.000 description 2
- 230000033764 rhythmic process Effects 0.000 description 2
- 238000012216 screening Methods 0.000 description 2
- 238000002560 therapeutic procedure Methods 0.000 description 2
- 206010047302 ventricular tachycardia Diseases 0.000 description 2
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 1
- 201000001320 Atherosclerosis Diseases 0.000 description 1
- 206010003662 Atrial flutter Diseases 0.000 description 1
- 206010003671 Atrioventricular Block Diseases 0.000 description 1
- 238000010453 CRISPR/Cas method Methods 0.000 description 1
- 206010007572 Cardiac hypertrophy Diseases 0.000 description 1
- 208000006029 Cardiomegaly Diseases 0.000 description 1
- 208000031229 Cardiomyopathies Diseases 0.000 description 1
- 102000053602 DNA Human genes 0.000 description 1
- 230000033616 DNA repair Effects 0.000 description 1
- 108091029865 Exogenous DNA Proteins 0.000 description 1
- 241000282326 Felis catus Species 0.000 description 1
- 206010064571 Gene mutation Diseases 0.000 description 1
- 206010019280 Heart failures Diseases 0.000 description 1
- 102000004310 Ion Channels Human genes 0.000 description 1
- 241000581650 Ivesia Species 0.000 description 1
- 101710163270 Nuclease Proteins 0.000 description 1
- 108091028043 Nucleic acid sequence Proteins 0.000 description 1
- 108700026244 Open Reading Frames Proteins 0.000 description 1
- 238000012408 PCR amplification Methods 0.000 description 1
- 108010016731 PPAR gamma Proteins 0.000 description 1
- 102000003728 Peroxisome Proliferator-Activated Receptors Human genes 0.000 description 1
- 108090000029 Peroxisome Proliferator-Activated Receptors Proteins 0.000 description 1
- 102000012132 Peroxisome proliferator-activated receptor gamma Human genes 0.000 description 1
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 1
- 206010042434 Sudden death Diseases 0.000 description 1
- 229920004890 Triton X-100 Polymers 0.000 description 1
- 239000013504 Triton X-100 Substances 0.000 description 1
- 230000002159 abnormal effect Effects 0.000 description 1
- 230000005856 abnormality Effects 0.000 description 1
- 230000004913 activation Effects 0.000 description 1
- 239000012190 activator Substances 0.000 description 1
- 125000003277 amino group Chemical group 0.000 description 1
- 238000002583 angiography Methods 0.000 description 1
- 239000003963 antioxidant agent Substances 0.000 description 1
- 230000003078 antioxidant effect Effects 0.000 description 1
- 210000001992 atrioventricular node Anatomy 0.000 description 1
- 230000001580 bacterial effect Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 210000000625 blastula Anatomy 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 230000023852 carbohydrate metabolic process Effects 0.000 description 1
- 230000000747 cardiac effect Effects 0.000 description 1
- 210000004413 cardiac myocyte Anatomy 0.000 description 1
- 238000013194 cardioversion Methods 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 210000000349 chromosome Anatomy 0.000 description 1
- 238000010367 cloning Methods 0.000 description 1
- 230000034994 death Effects 0.000 description 1
- 231100000517 death Toxicity 0.000 description 1
- 208000035475 disorder Diseases 0.000 description 1
- 230000005782 double-strand break Effects 0.000 description 1
- 230000034431 double-strand break repair via homologous recombination Effects 0.000 description 1
- 230000003828 downregulation Effects 0.000 description 1
- 238000002651 drug therapy Methods 0.000 description 1
- 230000037024 effective refractory period Effects 0.000 description 1
- 238000001962 electrophoresis Methods 0.000 description 1
- 210000001671 embryonic stem cell Anatomy 0.000 description 1
- 230000002996 emotional effect Effects 0.000 description 1
- 230000037149 energy metabolism Effects 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 210000003734 kidney Anatomy 0.000 description 1
- 238000002372 labelling Methods 0.000 description 1
- 230000037356 lipid metabolism Effects 0.000 description 1
- 210000001161 mammalian embryo Anatomy 0.000 description 1
- 239000003550 marker Substances 0.000 description 1
- 230000035800 maturation Effects 0.000 description 1
- 230000008558 metabolic pathway by substance Effects 0.000 description 1
- 238000010172 mouse model Methods 0.000 description 1
- 230000035772 mutation Effects 0.000 description 1
- 208000031225 myocardial ischemia Diseases 0.000 description 1
- 210000001087 myotubule Anatomy 0.000 description 1
- 239000002547 new drug Substances 0.000 description 1
- 210000004940 nucleus Anatomy 0.000 description 1
- 210000000056 organ Anatomy 0.000 description 1
- 210000003101 oviduct Anatomy 0.000 description 1
- 238000004806 packaging method and process Methods 0.000 description 1
- 230000008506 pathogenesis Effects 0.000 description 1
- 230000001575 pathological effect Effects 0.000 description 1
- 230000010412 perfusion Effects 0.000 description 1
- 108010060054 peroxisome-proliferator-activated receptor-gamma coactivator-1 Proteins 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 230000002035 prolonged effect Effects 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 238000000718 qrs complex Methods 0.000 description 1
- 230000008439 repair process Effects 0.000 description 1
- 238000012827 research and development Methods 0.000 description 1
- 238000010008 shearing Methods 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 239000008223 sterile water Substances 0.000 description 1
- 230000001954 sterilising effect Effects 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 238000001356 surgical procedure Methods 0.000 description 1
- 230000009885 systemic effect Effects 0.000 description 1
- 229960001603 tamoxifen Drugs 0.000 description 1
- 238000010809 targeting technique Methods 0.000 description 1
- 230000001225 therapeutic effect Effects 0.000 description 1
- 230000035924 thermogenesis Effects 0.000 description 1
- 229940035893 uracil Drugs 0.000 description 1
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
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
- C12N15/8509—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells for producing genetically modified animals, e.g. transgenic
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
- A01K67/027—New or modified breeds of vertebrates
- A01K67/0275—Genetically modified vertebrates, e.g. transgenic
- A01K67/0276—Knock-out vertebrates
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K49/00—Preparations for testing in vivo
- A61K49/0004—Screening or testing of compounds for diagnosis of disorders, assessment of conditions, e.g. renal clearance, gastric emptying, testing for diabetes, allergy, rheuma, pancreas functions
- A61K49/0008—Screening agents using (non-human) animal models or transgenic animal models or chimeric hosts, e.g. Alzheimer disease animal model, transgenic model for heart failure
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2217/00—Genetically modified animals
- A01K2217/15—Animals comprising multiple alterations of the genome, by transgenesis or homologous recombination, e.g. obtained by cross-breeding
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2227/00—Animals characterised by species
- A01K2227/10—Mammal
- A01K2227/105—Murine
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2267/00—Animals characterised by purpose
- A01K2267/03—Animal model, e.g. for test or diseases
- A01K2267/035—Animal model for multifactorial diseases
- A01K2267/0375—Animal model for cardiovascular diseases
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Zoology (AREA)
- Genetics & Genomics (AREA)
- Engineering & Computer Science (AREA)
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Veterinary Medicine (AREA)
- Biomedical Technology (AREA)
- Biotechnology (AREA)
- General Engineering & Computer Science (AREA)
- Toxicology (AREA)
- Gastroenterology & Hepatology (AREA)
- Wood Science & Technology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Molecular Biology (AREA)
- Biochemistry (AREA)
- Biophysics (AREA)
- Environmental Sciences (AREA)
- Animal Behavior & Ethology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Endocrinology (AREA)
- Physics & Mathematics (AREA)
- Plant Pathology (AREA)
- Microbiology (AREA)
- Medicinal Chemistry (AREA)
- Biodiversity & Conservation Biology (AREA)
- Diabetes (AREA)
- Animal Husbandry (AREA)
- Pathology (AREA)
- Rheumatology (AREA)
- Urology & Nephrology (AREA)
- Epidemiology (AREA)
- Public Health (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
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, and is one of the main causes of sudden cardiac death (sudden cardiac death, SCD). 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 (Homology directed repair, HDR), so that the effect of fragment knock-in or editing is achieved. 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 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 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 the DNA of the tail of the F1 generation mice, and obtaining the gene type PGC-1 alpha by PCR identification flox+/- Mating with wild type mice to obtain F2 generation mice; 3) PGC-1 alpha from F2 mice flox+/- Mice, PGC-1 alpha flox+/- The mice are mutually inbred to generate F3 generation to obtain stable PGC-1 alpha flox+/+ A strain of mice; 4) PGC-1 alpha flox+/+ Mice were hybridized with different kinds of cre mice to obtain 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; cKO region selects exon 4-5 of PGC-1 alpha gene of mouse model as cKO region, resulting in shift of downstream open reading frame of transcribed transcript, deleting this region will result in loss of function of PGC-1 alpha gene of mouse, achieving experimental effect of gene knockout, with low cost, high success rate and high practicality; 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 PGC-1 alpha containing Loxp locus based on CRISPR/Cas9 system flox+/+ Mice, mating with cre mice of different specificities, can give different tissue specificities or notThe model of arrhythmia is built by the same tissue-specific induced knockout PGC-1 alpha mice, so that not only can the cost be saved, but also convenience is provided 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 invention and are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description serve to explain the invention. 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 a 5 '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.
In fig. 4, 2, 3 and 4 represent F1-generation mouse samples, M represents DNA Marker, WT represents wild-type control sample, water represents Water blank control sample, 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 shows a myocardial specific knockout of PGC-1α (PGC-1α) according to the present invention flox+/+,myh6 ) Mouse identification results.
FIG. 10 is a schematic diagram of the propagation of mice according to the present invention.
FIG. 11 shows PGC-1 alpha according to the present invention flox+/+ Mice and PGC-1 alpha flox+/+,myh6 The ECG of the mice represents the map.
Wherein Control in the figure represents PGC-1α flox+/+ A mouse; model represents PGC-1 alpha flox+/+,myh6 And (3) a mouse.
FIG. 12 shows PGC-1 alpha according to the present invention flox+/+ Mice and PGC-1 alpha flox+/+,myh6 Ventricular conduction in mice represents the graph.
FIG. 13 shows PGC-1 alpha according to the present invention flox+/+ Mice and PGC-1 alpha flox+/+,myh6 The field potential of the mice represents the graph.
FIG. 14 shows PGC-1 alpha according to the present invention flox+/+ Mice and PGC-1 alpha flox+/+,myh6 Arrhythmia induction pattern in mice.
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 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 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 the DNA of the tail of the F1 generation mice, and obtaining the gene type PGC-1 alpha by PCR identification flox+/- Mating with wild type mice to obtain F2 generation mice; 3) PGC-1 alpha from F2 mice flox+/- Mice, PGC-1 alpha flox+/- The mice are mutually inbred to generate F3 generation to obtain stable PGC-1 alpha flox+/+ A strain of mice; 4) PGC-1 alpha flox+/+ Mice were hybridized with different kinds of cre mice to obtain 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 to 5 were selected as conditional knockdown regions (cKO region). Deletion of this region results in deletion of the mouse Ppargc1a (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 the on-line design software CRISPOR (http:// CRISPOR. Tefor. Net), 2 specific sites were selected as target sequences of gRNA within target sites exon 4 and 5 of the mouse PGC-1α Gene, respectively: reverse strand (reverse 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 Homology arm (Homology arm) and conditional knockout (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,2 XClonExpress Mix 5. Mu.L (Norpran, 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
Cas9mRNA, 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. Fertilized eggs continue to develop into individuals, and the mice are identified with the rat tail 7 days after birth to obtain positive F0 generation Foundator mice (the identification method is the same as the following 4, and the genotype is PGC-1 alpha flox+/- Identification and screening of 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 Cas9mRNA, 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 0.8uL 100pmol/uL CrRNA into 5.2uL RNase-free water (RNase-free water), adding 0.6uL 100pmol/uL TracRNA, mixing, incubating for 5min, and adding 0.2uL 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;
among them, tracRNA (Integrated DNA Technologies company, IDT), sequence:
5'-AAACAGCAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCU-3' (SEQ ID NO:10, if expressed as ST.26, uracil "U" in the sequence is represented as "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. Genotype PGC-1 alpha flox+/- Identification and screening of mice
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 length 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 |
ddH 2 O | 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) F1 generation PGC-1 alpha flox+/- Identification result
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 correct size and position of the PCR product fragments of 3F 1 mice can be determined that the genotypes of the 3F 1 mice are 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: wild-type mouse fragment size 7.16kb, PGC-1 alpha flox+/- Fragments of 7.16kb and 5.41kb in size, mouse PGC-1. Alpha flox+/+ The mouse fragment was 5.41kb in size. And (3) probe labeling amplified by PCR (polymerase chain reaction) of a 3' -end probe designed after BstEII enzyme digestion: wild-type mouse fragment size 13.73kb, PGC-1. Alpha flox+/- Fragment sizes were 13.73kb and 5.84kb, PGC-1. Alpha flox+/+ The mouse fragment was 5.84kb in size.
F1 generation mice 2, 3 and 4 are PGC-1 alpha flox+/- Mice, therefore, had a Southern blot result of a 5'probe-SacI fragment size of 7.16kb and a 5.41kb fragment, and a 3' probe-BstEII fragment of 13.73kb and a 5.84kb fragment.
5. Propagation and Gene identification
(1) F3 generation PGC-1 alpha flox+/+ Mouse identification:
f1 generation PGC-1 alpha flox+/- The mice are verified to be free of problems, and the primers can be designed for loxp sites to identify PGC-1 alpha flox+/+ And (3) mice. 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, and PGC-1 alpha is selected from the F2-generation mice flox+/- The composition of the mice and the method for preparing the same,PGC-1α flox+/- the mice are mutually inbred to generate F3 generation to obtain stable PGC-1 alpha flox+/+ Mice strain.
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. PGC-1 alpha flox+/+ The size of the flox fragment is 250bp, and myh is not expressed. PGC-1 alpha flox+/+,myh6 The flox fragment size in the mice was 250bp and the myh6 fragment size 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 knockouts. 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
By PGC-1 alpha flox+/+ Hybridization of mice with Myh6-cre (aMHC-MerCreMer) mice to give PGC-1 a flox+/+,myh6 And (3) mice. For 8-10 week old male PGC-1 alpha flox+/+ Mice (Control group) and PGC-1 alpha flox+/+,myh6 Mice (Model group) were continuously injected with tamoxifen (40 mg/kg) for 5 days. After the last administration for 4 weeks, the isolated heart of the mouse is hung on a Langendorff 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 left ventricle and right using 128 channel electrical mappingVentricles, recording sinus and field potential signals under 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 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 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 is located at 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 the DNA of the tail of the F1 generation mice, and obtaining the gene type PGC-1 alpha by PCR identification flox+/- Mating with wild type mice to obtain F2 generation mice;
3) PGC-1 alpha from F2 mice flox+/- Mice, PGC-1 alpha flox+/- The mice are mutually inbred to generate F3 generation to obtain stable PGC-1 alpha flox+/+ A strain of mice;
4)PGC-1α flox+/+ mice were hybridized with different kinds of cre mice to obtain 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.
10. Use of a PGC-1 alpha knockout mouse animal model obtained by the method of any one of claims 1-8 in the manufacture of an antiarrhythmic medicament.
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202311205731.5A CN117568398B (en) | 2023-09-19 | Method for constructing arrhythmia animal model by PGC-1 alpha gene knockout mice and application thereof |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202311205731.5A CN117568398B (en) | 2023-09-19 | Method for constructing arrhythmia animal model by PGC-1 alpha gene knockout mice and application thereof |
Publications (2)
Publication Number | Publication Date |
---|---|
CN117568398A true CN117568398A (en) | 2024-02-20 |
CN117568398B CN117568398B (en) | 2024-06-04 |
Family
ID=
Citations (9)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20030124598A1 (en) * | 2001-11-09 | 2003-07-03 | Dana-Farber Cancer Institute, Inc. | PGC-1beta, a novel PGC-1 homologue and uses therefor |
JP2010115125A (en) * | 2008-11-11 | 2010-05-27 | Mie Univ | Method for screening metabolic activator, new metabolic activator and method for producing hypermetabolic model mouse |
CN102051380A (en) * | 2009-10-29 | 2011-05-11 | 哈尔滨医科大学 | Method for establishing arrhythmia animal model |
WO2016187717A1 (en) * | 2015-05-26 | 2016-12-01 | Exerkine Corporation | Exosomes useful for genome editing |
CN111909958A (en) * | 2020-07-09 | 2020-11-10 | 西安医学院 | Construction of mouse model of vascular smooth muscle cell conditional knockout Yap1 gene |
CN112980846A (en) * | 2021-04-09 | 2021-06-18 | 山西省人民医院 | Construction method of Pax2 conditional gene knockout mouse model |
US20220163511A1 (en) * | 2020-10-16 | 2022-05-26 | Brown University | Human in vitro cardiotoxicity model |
CN115399293A (en) * | 2022-08-17 | 2022-11-29 | 山东中医药大学 | Method for constructing arrhythmic cardiomyopathy animal model |
CN116019063A (en) * | 2022-10-21 | 2023-04-28 | 江南大学 | Construction method and application of mouse conditional induction Hmox1 gene knockout model |
Patent Citations (9)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20030124598A1 (en) * | 2001-11-09 | 2003-07-03 | Dana-Farber Cancer Institute, Inc. | PGC-1beta, a novel PGC-1 homologue and uses therefor |
JP2010115125A (en) * | 2008-11-11 | 2010-05-27 | Mie Univ | Method for screening metabolic activator, new metabolic activator and method for producing hypermetabolic model mouse |
CN102051380A (en) * | 2009-10-29 | 2011-05-11 | 哈尔滨医科大学 | Method for establishing arrhythmia animal model |
WO2016187717A1 (en) * | 2015-05-26 | 2016-12-01 | Exerkine Corporation | Exosomes useful for genome editing |
CN111909958A (en) * | 2020-07-09 | 2020-11-10 | 西安医学院 | Construction of mouse model of vascular smooth muscle cell conditional knockout Yap1 gene |
US20220163511A1 (en) * | 2020-10-16 | 2022-05-26 | Brown University | Human in vitro cardiotoxicity model |
CN112980846A (en) * | 2021-04-09 | 2021-06-18 | 山西省人民医院 | Construction method of Pax2 conditional gene knockout mouse model |
CN115399293A (en) * | 2022-08-17 | 2022-11-29 | 山东中医药大学 | Method for constructing arrhythmic cardiomyopathy animal model |
CN116019063A (en) * | 2022-10-21 | 2023-04-28 | 江南大学 | Construction method and application of mouse conditional induction Hmox1 gene knockout model |
Non-Patent Citations (14)
Title |
---|
KHALIL SAADEH等: "Molecular basis of ventricular arrhythmogenicity in a Pgc-1α deficient murine model", 《MOLECULAR GENETICS AND METABOLISM REPORTS》, 9 April 2021 (2021-04-09), pages 1 - 10 * |
LIU, B.等: "Mus musculus peroxisome proliferative activated receptor, gamma, coactivator 1 alpha (Ppargc1a), transcript variant 4, mRNA", 《GENBANK DATABASE》, 10 September 2023 (2023-09-10), pages 001402987 * |
SWAGOTA D ROY: "New mutant alleles for Spargel/dPGC-1 highlights the function of Spargel RRM domain in oogenesis and expands the role of Spargel in embryogenesis and intracellular transport", 《G3 (BETHESDA) 》, 30 August 2023 (2023-08-30) * |
TERESA C LEONE等: "PGC-1alpha deficiency causes multi-system energy metabolic derangements: muscle dysfunction, abnormal weight control and hepatic steatosis", 《PLOS BIOLOGY.》, 15 March 2005 (2005-03-15), pages 0672 - 0687 * |
侯婷婷;刘广忠;陶源;赵菁菁;李为民;: "心房颤动与心房重构研究的最新进展", 现代生物医学进展, no. 35, 20 December 2017 (2017-12-20), pages 182 - 185 * |
姜扬: "γ-氨基丁酸能神经元条件性敲除PGC-1α基因对小鼠焦虑抑郁样行为的影响", 《万方》, 27 March 2019 (2019-03-27), pages 1 - 76 * |
张城林;张艳;吴立玲;张福春;: "运动训练对心肌梗死后心脏的保护作用", 生理科学进展, no. 02, 25 April 2015 (2015-04-25), pages 59 - 64 * |
张梓桑;张薪茹;张庄;李光宇;张劲松;杜荣增;: "心房颤动患者外周血淋巴细胞β_3肾上腺素受体的表达水平及临床意义", 中国循环杂志, no. 04, 24 April 2019 (2019-04-24), pages 59 - 65 * |
张燕;王强;王东进;项阳;张辰宇;: "能量代谢与疾病的关键调控因子PGC-1α(下)", 中国糖尿病杂志, no. 08, 20 August 2008 (2008-08-20), pages 10 - 15 * |
樊翀: "PGC-1α基因rs8192678单核苷酸多态性与高血压、非瓣膜性心房纤颤的相关性研究", 《万方》, 5 April 2017 (2017-04-05), pages 1 - 55 * |
田静朴;王小兵;李为民;: "β3肾上腺素能受体与心房颤动心肌代谢重构的研究进展", 心脏杂志, no. 05, pages 120 - 123 * |
胡雪苹;郑天鹏;: "PGC-1α在糖尿病各类并发症的发病作用", 世界最新医学信息文摘, no. 92, 17 November 2017 (2017-11-17), pages 32 - 33 * |
郭茜;郭家彬;李梨;彭双清;: "PGC-1α与线粒体O生成调控在心血管疾病中的作用", 中国药理学通报, no. 01, 25 December 2012 (2012-12-25), pages 6 - 10 * |
韩劲松;阎德民;汪曾炜;朱洪玉;: "过氧化物酶体增殖物激活受体γ协同刺激因子1α在早期缺血预处理中的作用", 中国胸心血管外科临床杂志, no. 04, 25 August 2008 (2008-08-25), pages 59 - 64 * |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
CN108660161B (en) | Method for preparing chimeric gene-free knockout animal based on CRISPR/Cas9 technology | |
CN109628454B (en) | Construction method of zebra fish glycogen storage disease gys1 and gys2 gene mutant | |
CN106282231B (en) | Construction method and application of mucopolysaccharide storage disease type II animal model | |
CN111019971A (en) | Construction method of mouse model for conditionally overexpressing HPV E6 gene at ROSA26 site | |
CN112011538B (en) | Method for constructing Mutyh gene conditional knockout mouse model | |
CN110257435B (en) | Construction method and application of PROM1-KO mouse model | |
CN112029765A (en) | Method for making Metrnl gene conditional knockout mouse model | |
CN104611368A (en) | Carrier incapable of generating frameshift mutation after recombination as well as method and application for gene fixe-point knock-in in Xenopus laevis genome | |
CN112715483B (en) | Preparation method and application method of mutant CNPase zebra fish model for reducing cardiac function | |
CN112899311B (en) | Construction method and application of RS1-KO mouse model | |
CN111206054B (en) | Construction method of animal model for conditionally knocking out liver HO-1 gene by using CRISPR-Cas9 | |
CN111304258A (en) | Ndufs2 gene conditional point mutation mouse model and construction method and application thereof | |
CN113088521A (en) | Construction method of Ahnak2 gene knockout animal model based on CRISPR/Cas9 technology | |
CN108949832A (en) | A kind of targeting vector and its application for knock-out pig GHR gene | |
CN117568398B (en) | Method for constructing arrhythmia animal model by PGC-1 alpha gene knockout mice and application thereof | |
CN114457114B (en) | Construction method of animal model for conditional knockout of Fars2 gene | |
CN114480497B (en) | Construction and application method of ep400 gene knockout zebra fish heart failure model | |
CN111778278A (en) | Construction method and application of Slfn 4-deleted atherosclerosis model mouse | |
CN114591957B (en) | Construction method and application of severe hemophilia A animal model | |
Mademtzoglou et al. | A p57 conditional mutant allele that allows tracking of p57‐expressing cells | |
CN117568398A (en) | Method for constructing arrhythmia animal model by PGC-1 alpha gene knockout mice and application thereof | |
CN113897399A (en) | Scn1lab gene knockout zebra fish epilepsy model and application thereof | |
CN113897361A (en) | Eef1b2 gene knockout zebra fish epilepsy model and construction method and application thereof | |
CN113957070A (en) | Chd2 gene knockout zebra fish epilepsy model and construction method and application thereof | |
CN113897362A (en) | Scn1lab gene knockout zebra fish epilepsy model and construction method and application thereof |
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 |