CN112522312A - WKH rat model construction method - Google Patents
WKH rat model construction method Download PDFInfo
- Publication number
- CN112522312A CN112522312A CN202011326345.8A CN202011326345A CN112522312A CN 112522312 A CN112522312 A CN 112522312A CN 202011326345 A CN202011326345 A CN 202011326345A CN 112522312 A CN112522312 A CN 112522312A
- Authority
- CN
- China
- Prior art keywords
- wkh
- gene
- rat model
- hypertension
- mouse
- 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
- 238000011552 rat model Methods 0.000 title claims abstract description 31
- 238000010276 construction Methods 0.000 title claims description 10
- 206010020772 Hypertension Diseases 0.000 claims abstract description 55
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 55
- 108091033409 CRISPR Proteins 0.000 claims abstract description 23
- 241001465754 Metazoa Species 0.000 claims abstract description 23
- 238000000034 method Methods 0.000 claims abstract description 20
- 238000005516 engineering process Methods 0.000 claims abstract description 19
- 238000010354 CRISPR gene editing Methods 0.000 claims abstract description 10
- 230000036772 blood pressure Effects 0.000 claims abstract description 10
- 230000001717 pathogenic effect Effects 0.000 claims abstract description 10
- 102000004169 proteins and genes Human genes 0.000 claims abstract description 5
- 238000012163 sequencing technique Methods 0.000 claims abstract description 5
- 230000035488 systolic blood pressure Effects 0.000 claims abstract description 5
- 108091026890 Coding region Proteins 0.000 claims abstract description 4
- 230000002776 aggregation Effects 0.000 claims abstract description 4
- 238000004220 aggregation Methods 0.000 claims abstract description 4
- 230000007918 pathogenicity Effects 0.000 claims abstract description 4
- 241000700159 Rattus Species 0.000 claims description 40
- 241000699666 Mus <mouse, genus> Species 0.000 claims description 18
- 238000010171 animal model Methods 0.000 claims description 9
- 230000035772 mutation Effects 0.000 claims description 8
- 241000283973 Oryctolagus cuniculus Species 0.000 claims description 6
- 235000013305 food Nutrition 0.000 claims description 6
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 6
- 238000003975 animal breeding Methods 0.000 claims description 4
- 238000009395 breeding Methods 0.000 claims description 4
- 230000001488 breeding effect Effects 0.000 claims description 4
- 238000001514 detection method Methods 0.000 claims description 3
- 238000011160 research Methods 0.000 abstract description 8
- 238000011161 development Methods 0.000 abstract description 6
- 230000008506 pathogenesis Effects 0.000 abstract description 4
- 230000003276 anti-hypertensive effect Effects 0.000 abstract description 3
- 101000867817 Homo sapiens Voltage-dependent L-type calcium channel subunit alpha-1D Proteins 0.000 description 22
- 102000014816 CACNA1D Human genes 0.000 description 14
- 230000001631 hypertensive effect Effects 0.000 description 10
- 241000699670 Mus sp. Species 0.000 description 9
- 206010064571 Gene mutation Diseases 0.000 description 8
- 230000008859 change Effects 0.000 description 6
- 238000010356 CRISPR-Cas9 genome editing Methods 0.000 description 4
- 108091034117 Oligonucleotide Proteins 0.000 description 4
- 230000036962 time dependent Effects 0.000 description 4
- 101150025500 CACNA1D gene Proteins 0.000 description 3
- 229940127291 Calcium channel antagonist Drugs 0.000 description 3
- 108020005004 Guide RNA Proteins 0.000 description 3
- 102000004016 L-Type Calcium Channels Human genes 0.000 description 3
- 108090000420 L-Type Calcium Channels Proteins 0.000 description 3
- 101000836074 Mus musculus Prostatic spermine-binding protein Proteins 0.000 description 3
- 101000703431 Mus musculus Sex hormone-binding globulin Proteins 0.000 description 3
- 102100032575 Voltage-dependent L-type calcium channel subunit alpha-1D Human genes 0.000 description 3
- 230000009471 action Effects 0.000 description 3
- 208000015345 genetic hypertension Diseases 0.000 description 3
- 102100033902 Endothelin-1 Human genes 0.000 description 2
- 101800004490 Endothelin-1 Proteins 0.000 description 2
- 101100058876 Rattus norvegicus Cacna1d gene Proteins 0.000 description 2
- 238000012300 Sequence Analysis Methods 0.000 description 2
- JLCPHMBAVCMARE-UHFFFAOYSA-N [3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-[[3-[[3-[[3-[[3-[[3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-hydroxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methyl [5-(6-aminopurin-9-yl)-2-(hydroxymethyl)oxolan-3-yl] hydrogen phosphate Polymers Cc1cn(C2CC(OP(O)(=O)OCC3OC(CC3OP(O)(=O)OCC3OC(CC3O)n3cnc4c3nc(N)[nH]c4=O)n3cnc4c3nc(N)[nH]c4=O)C(COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3CO)n3cnc4c(N)ncnc34)n3ccc(N)nc3=O)n3cnc4c(N)ncnc34)n3ccc(N)nc3=O)n3ccc(N)nc3=O)n3ccc(N)nc3=O)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cc(C)c(=O)[nH]c3=O)n3cc(C)c(=O)[nH]c3=O)n3ccc(N)nc3=O)n3cc(C)c(=O)[nH]c3=O)n3cnc4c3nc(N)[nH]c4=O)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)O2)c(=O)[nH]c1=O JLCPHMBAVCMARE-UHFFFAOYSA-N 0.000 description 2
- 210000000709 aorta Anatomy 0.000 description 2
- 210000002565 arteriole Anatomy 0.000 description 2
- 210000001367 artery Anatomy 0.000 description 2
- 239000000480 calcium channel blocker Substances 0.000 description 2
- 230000008602 contraction Effects 0.000 description 2
- 230000034431 double-strand break repair via homologous recombination Effects 0.000 description 2
- 230000035622 drinking Effects 0.000 description 2
- 229940079593 drug Drugs 0.000 description 2
- 239000003814 drug Substances 0.000 description 2
- 235000013601 eggs Nutrition 0.000 description 2
- 238000010353 genetic engineering Methods 0.000 description 2
- 238000011700 genetic hypertension animal model Methods 0.000 description 2
- 238000010362 genome editing Methods 0.000 description 2
- 210000002216 heart Anatomy 0.000 description 2
- 102000053844 human CACNA1D Human genes 0.000 description 2
- 230000006872 improvement Effects 0.000 description 2
- 210000003734 kidney Anatomy 0.000 description 2
- 108020004999 messenger RNA Proteins 0.000 description 2
- 229960001597 nifedipine Drugs 0.000 description 2
- HYIMSNHJOBLJNT-UHFFFAOYSA-N nifedipine Chemical compound COC(=O)C1=C(C)NC(C)=C(C(=O)OC)C1C1=CC=CC=C1[N+]([O-])=O HYIMSNHJOBLJNT-UHFFFAOYSA-N 0.000 description 2
- 238000005457 optimization Methods 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- 238000012216 screening Methods 0.000 description 2
- 230000037432 silent mutation Effects 0.000 description 2
- 230000008685 targeting Effects 0.000 description 2
- 230000009261 transgenic effect Effects 0.000 description 2
- YNGDWRXWKFWCJY-UHFFFAOYSA-N 1,4-Dihydropyridine Chemical compound C1C=CNC=C1 YNGDWRXWKFWCJY-UHFFFAOYSA-N 0.000 description 1
- 108091032955 Bacterial small RNA Proteins 0.000 description 1
- 241001260012 Bursa Species 0.000 description 1
- 102000003922 Calcium Channels Human genes 0.000 description 1
- 108090000312 Calcium Channels Proteins 0.000 description 1
- 208000024172 Cardiovascular disease Diseases 0.000 description 1
- 206010010356 Congenital anomaly Diseases 0.000 description 1
- 230000005778 DNA damage Effects 0.000 description 1
- 231100000277 DNA damage Toxicity 0.000 description 1
- 108700024394 Exon Proteins 0.000 description 1
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 1
- 208000001953 Hypotension Diseases 0.000 description 1
- 101710163270 Nuclease Proteins 0.000 description 1
- 108091081024 Start codon Proteins 0.000 description 1
- 108020005038 Terminator Codon Proteins 0.000 description 1
- 230000002159 abnormal effect Effects 0.000 description 1
- 230000005856 abnormality Effects 0.000 description 1
- 239000002220 antihypertensive agent Substances 0.000 description 1
- 229940127088 antihypertensive drug Drugs 0.000 description 1
- 238000013459 approach Methods 0.000 description 1
- 230000004872 arterial blood pressure Effects 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 210000004413 cardiac myocyte Anatomy 0.000 description 1
- 210000004027 cell Anatomy 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 210000000349 chromosome Anatomy 0.000 description 1
- 230000027288 circadian rhythm Effects 0.000 description 1
- 238000003776 cleavage reaction Methods 0.000 description 1
- 238000009404 close breeding Methods 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 230000007812 deficiency Effects 0.000 description 1
- 230000035487 diastolic blood pressure Effects 0.000 description 1
- 230000037213 diet Effects 0.000 description 1
- 235000005911 diet Nutrition 0.000 description 1
- 125000004925 dihydropyridyl group Chemical group N1(CC=CC=C1)* 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 239000003651 drinking water Substances 0.000 description 1
- 235000020188 drinking water Nutrition 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 210000003038 endothelium Anatomy 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 238000010363 gene targeting Methods 0.000 description 1
- 230000002068 genetic effect Effects 0.000 description 1
- 230000007614 genetic variation Effects 0.000 description 1
- 210000005003 heart tissue Anatomy 0.000 description 1
- 208000021822 hypotensive Diseases 0.000 description 1
- 230000001077 hypotensive effect Effects 0.000 description 1
- 230000036737 immune function Effects 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 230000013011 mating Effects 0.000 description 1
- 210000000713 mesentery Anatomy 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000007170 pathology Effects 0.000 description 1
- 230000004796 pathophysiological change Effects 0.000 description 1
- 230000035778 pathophysiological process Effects 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 230000002265 prevention Effects 0.000 description 1
- 230000001603 reducing effect Effects 0.000 description 1
- 230000008439 repair process Effects 0.000 description 1
- 230000033764 rhythmic process Effects 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- 210000002966 serum Anatomy 0.000 description 1
- 238000010008 shearing Methods 0.000 description 1
- 230000003007 single stranded DNA break Effects 0.000 description 1
- 230000002269 spontaneous effect Effects 0.000 description 1
- 238000010186 staining Methods 0.000 description 1
- 230000001225 therapeutic effect Effects 0.000 description 1
- 230000008719 thickening Effects 0.000 description 1
- 210000001685 thyroid gland Anatomy 0.000 description 1
- 210000001519 tissue Anatomy 0.000 description 1
- 238000013518 transcription Methods 0.000 description 1
- 230000035897 transcription Effects 0.000 description 1
- 210000005239 tubule Anatomy 0.000 description 1
- 239000005526 vasoconstrictor agent Substances 0.000 description 1
- 239000013598 vector Substances 0.000 description 1
- 230000003442 weekly effect Effects 0.000 description 1
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—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
-
- 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
-
- 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
- C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
- C12N15/102—Mutagenizing nucleic acids
-
- 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
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
- C12N9/22—Ribonucleases RNAses, DNAses
-
- 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
- A01K2207/00—Modified animals
- A01K2207/05—Animals modified by non-integrating nucleic acids, e.g. antisense, RNAi, morpholino, episomal vector, for non-therapeutic purpose
-
- 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
- A01K2227/00—Animals characterised by species
- A01K2227/10—Mammal
- A01K2227/107—Rabbit
-
- 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
-
- 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
- C12N2800/00—Nucleic acids vectors
- C12N2800/10—Plasmid DNA
- C12N2800/106—Plasmid DNA for vertebrates
- C12N2800/107—Plasmid DNA for vertebrates for mammalian
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Genetics & Genomics (AREA)
- Engineering & Computer Science (AREA)
- Organic Chemistry (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Biotechnology (AREA)
- General Engineering & Computer Science (AREA)
- Biomedical Technology (AREA)
- General Health & Medical Sciences (AREA)
- Molecular Biology (AREA)
- Biochemistry (AREA)
- Biophysics (AREA)
- Microbiology (AREA)
- Veterinary Medicine (AREA)
- Medicinal Chemistry (AREA)
- Environmental Sciences (AREA)
- Physics & Mathematics (AREA)
- Plant Pathology (AREA)
- Toxicology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Gastroenterology & Hepatology (AREA)
- Animal Behavior & Ethology (AREA)
- Animal Husbandry (AREA)
- Biodiversity & Conservation Biology (AREA)
- Crystallography & Structural Chemistry (AREA)
- Investigating Or Analysing Biological Materials (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention relates to the technical field of a method for constructing a hereditary hypertension rat model, in particular to a method for constructing a WKH rat model, which comprises the following steps: adopting gene sequencing technology to detect coding regions and flanking regions of 43 pathogenic genes related to single-gene hypertension of clinical teenager hypertension patients with familial aggregation, predicting the influence of newly-discovered mutant genes on protein structure and function through a plurality of software, and determining the pathogenic genes through pathogenicity judgment; then, the WKH rat model is constructed by utilizing the gene engineering CRISPR/Cas9 technology, the postnatal blood pressure of the mutant gene animal is continuously increased along with the age of the animal, and the systolic pressure of most offspring tail animals is more than 140 mmHg. Provides a research tool for the function of the gene in the development of hypertension, and provides a research model for continuously exploring the pathogenesis related to hypertension and exploring a new target point of the antihypertensive effect.
Description
Technical Field
The invention relates to the technical field of a method for constructing a hereditary hypertension rat model, in particular to a method for constructing a WKH rat model, namely a method for constructing a humanized hereditary hypertension rat model.
Background
For the research of human hypertension pathogenesis and prevention and treatment scheme, a genetic hypertension model can be constructed by a genetic method, such as a selective recent propagation hypertension model and a genetic engineering hypertension model. The former model is to perform close breeding of the screened congenital hypertension animal for a plurality of generations (more than 20 generations) to obtain stable hypertension inheritance. Hypertensive rabbits, rats and mice have been developed. Rats are commonly used, and more mature varieties include: spontaneous Hypertensive Rats (SHR), Dahl salt-sensitive rats (DS), Milan hypertensive rats (MHS), and hereditary hypertensive rats (GH) are also called New Zealand hypertensive rats, Israel hypertensive rats (SBH), and Lyon hypertensive rats (LH). Although these models are very similar to human hypertension, the following deficiencies still exist: it is obtained through selective reproduction and is different from human diseases. ② thyroid and immune function are abnormal.
Genetically engineered hypertension models can be divided into two categories: one is to adopt transgenic technology to integrate exogenous genes into animal genome at random and over-express to cause hypertension, so called hypertension transgenic animal; another is the targeted knockout of endogenous genes in animals using gene targeting techniques to cause hypertension, which is called hypertensive knockout animals. Because the technology and laboratory requirements for preparing the gene engineering hypertension model are higher, the reports of successfully preparing the gene engineering hypertension model in China are less. With the development of CRISPR-Cas9 technology (a gene manipulation tool), it has recently been widely used in various experimental models such as cell lines, laboratory animals, plants, and even human clinical trials. The CRISPR-Cas9 system includes a guide Cas9 nuclease, guided by a small RNA molecule, creating site-directed double-stranded DNA breaks. A process that allows permanent modification of a genomic target sequence can repair DNA damage. The application of the system is proposed to provide a new approach for treating hypertension and cardiovascular diseases. Therefore, the animal model aims to screen pathogenic genes from hypertension families, a specific genetic variation genetic hypertension rat model (WKH rat model) is constructed by using a CRISPR-Cas9 gene editing technology, and a new tool is provided for screening antihypertensive drugs with specific action targets.
Cav1.3L-type calcium channel alpha 1 subunit coding gene CACNA1D is related to hypertension, the literature reports that CACNA1D gene is a newly found blood pressure susceptible region, Chinese scholars find that rs9810888 polymorphism is obviously related to diastolic pressure and average arterial pressure, poor living mode and rs9810888 GG genotype cooperation can influence blood pressure level of Chinese teenagers, and the change of CACNA1D gene function can influence the blood pressure reducing effect of a calcium channel blocker.
Disclosure of Invention
The invention aims to provide a WKH rat model construction method, which overcomes the defects of the prior art, provides a research tool for the action in the development of hypertension, and provides a research model for continuously exploring the pathogenesis related to hypertension and exploring a new target point of the antihypertensive action.
One of the technical schemes of the invention is realized by the following measures: a method for constructing a WKH rat model comprises the following steps: adopting gene sequencing technology to detect coding regions and flanking regions of 43 pathogenic genes related to single-gene hypertension of clinical teenager hypertension patients with familial aggregation, predicting the influence of newly-discovered mutant genes on protein structure and function through a plurality of software, and determining the pathogenic genes through pathogenicity judgment; then, the WKH rat model is constructed by utilizing the gene engineering CRISPR/Cas9 technology, the postnatal blood pressure of the mutant gene animal is continuously increased along with the age of the animal, and the systolic pressure of most offspring tail animals is more than 140 mmHg.
The following is a further optimization or/and improvement of one of the above-mentioned technical solutions of the invention:
the mutant gene is the gene detection result of a special type of hypertension patient.
The experimental animal is selected from a mouse, a rat or a rabbit.
The specific gene point mutation F0 mouse is constructed by CRISPR/Cas9 technology.
The specific gene mutation mouse is a descendant bred by the constructed F0 mouse.
The breeding condition of the mutant mice is an SPF animal breeding room, the room temperature is kept at 22 +/-2 ℃, the day and night period is 12 hours, and the mutant mice can be fed with food and water freely.
The invention provides a construction method of a human Cav1.3 hereditary hypertension rat model (Wang-Kang-Hui, WKH rat model). Firstly, discovering a new nosable Cav1.3 gene mutation site of hypertension clinically, determining a research target gene through document retrieval and software prediction, constructing and screening a specific Cav1.3 gene mutation (WKH) rat model by utilizing a gene engineering CRISPR/Cas9 technology, wherein compared with a wild type WKH rat, the screened WKH specific single-gene mutation rat starts to increase the blood pressure from the age of 6 months, the volume of glomeruli is increased at the age of 8 months, contraction occurs, the renal bursa is swollen, the renal tubules are expanded, the lumen structure is unclear, the wall of the renal arteriole is thickened, and the fibrous tissue is increased; cardiomyocyte enlargement; secondary artery thickening of mesentery, endothelium contraction and relaxation function abnormality and other pathophysiological changes; the progress of these pathologies can be prevented or alleviated by therapeutic measures such as hypotensive drugs; when the Cav1.3 gene is further hybridized with WKH rats with two site variations, hypertension is completely developed in three months. The similarity protein structure search based on NCBI blasts shows that these Cav1.3 gene variation sites can be homologously modeled. In a word, the WKH rat model has specific humanized mutation, can provide a research tool for the effect of the gene in the development of hypertension, and provides a research model for continuously exploring pathogenesis related to hypertension and exploring a new target point of the antihypertensive effect.
Drawings
FIG. 1 shows the comparison of the 7-32 week systolic blood pressure (SBP, 6 in each group) of WKH wild type SD rat and WKH gene heterozygous variation and homozygous variation:*P<0.05 and**P<0.01: heterozygous mouse and wild typeCompared with the mouse, the mouse is added with the compound,#P<0.05: homozygous mice were compared to heterozygous mice. A, a CACNA1D p.307 locus male mouse SBP time-varying trend graph; b: a trend graph of time-dependent change of SBP of female mouse at CACNA1D p.307 locus; c: trend graph of male mouse SBP of CACNA1D p.936 locus with time; d: time-dependent trend chart of female mouse SBP at CACNA1D p.936 locus; e: a trend graph of the change of the SBP of the male mouse at the position of CACNA1D p.1516 along with time; f: trend graph of time-dependent change of SBP of female mouse with CACNA1D p.1516 locus; g: a trend graph of the change of the SBP of the male mice of the single gene heterozygous site variation of the CACNA1D p.307 site, the CACNA1D p.936 site and the combined variation of the two sites of the single gene heterozygous site variation with time; h: time-dependent trend chart of single-site heterozygous variation of CACNA1D p.307 site and CACNA1D p.1516 site and combined variation of two sites of the single-site heterozygous variation of the SBP of the male mouse.
FIG. 2 shows the result of detecting endothelin-1 as a plasma vasoconstrictor in 6-month WKY male rats (6 rats in each group) with different genotypes of ACNA1D p.307: *: compared with wild mouseP<0.05。
FIGS. 3, 4 and 5 show the change in kidney histomorphology of different genotypes of 8 month CACNA1D p.307 WKH rats, respectively.
FIG. 6 shows EH staining comparisons of mesenteric secondary arterioles of WKH rats of different genotypes (AA, AG and GG genotypes from left to right).
FIG. 7 shows the morphology and statistics of heart tissues of different genotypes of 8-month-old WKH rats.
FIG. 8 shows the positions of the CACNA1D p.307, CACNA1D p.936 and CACNA1D p.1516 sites in the L-type calcium channel cav1.3, B: action position of dihydropyridine calcium channel antagonist nifedipine in L-type calcium channel cav1.3, C: non-dihydropyridine nifedipine calcium channel antagonists act at the site of the L-type calcium channel cav 1.3.
Detailed Description
In the first embodiment, the WKH rat model construction method is carried out according to the following steps: adopting gene sequencing technology to detect coding regions and flanking regions of 43 pathogenic genes related to single-gene hypertension of clinical teenager hypertension patients with familial aggregation, predicting the influence of newly-discovered mutant genes on protein structure and function through a plurality of software, and determining the pathogenic genes through pathogenicity judgment; then, the WKH rat model is constructed by utilizing the gene engineering CRISPR/Cas9 technology, the postnatal blood pressure of the mutant gene animal is continuously increased along with the age of the animal, and the systolic pressure of most offspring tail animals is more than 140 mmHg.
The following is a further optimization or/and improvement of the first embodiment:
the mutant gene is the gene detection result of a special type of hypertension patient.
The experimental animal is selected from mouse, rat or rabbit.
The specific gene point mutation F0 mouse is constructed by CRISPR/Cas9 technology.
The specific gene mutation mouse is the offspring bred by the constructed F0 mouse.
The breeding conditions of the mutant mice are SPF animal breeding rooms, the room temperature is kept at 22 +/-2 ℃, the day and night period is 12 hours, and the mutant mice can be freely fed and drunk.
In a second embodiment, the method for constructing the WKH rat model comprises the following steps: selecting a pathogenic gene of a hypertensive, and constructing the humanized genetic hypertension animal model by using a genetic engineering CRISPR/Cas9 technology.
After the model is established, the experimental animal is kept in daily feeding, and the genotype of the model animal is determined by a PCR method through tail shearing.
Heterozygous and homozygous mutant genotypes are selected from the experimental animals, and individuals with the blood pressure of the tail artery of more than 140mmHg are determined to be used as the human genetic hypertension model by using a BP-98A intelligent noninvasive sphygmomanometer mouse.
The experimental animal is selected from a mouse, a rat or a rabbit, and all experimental animals are superior to SD rats.
The influence of human Cav1.3 gene mutation factors on the development of hypertension is simulated, and the process is closer to the human hypertension pathophysiological process.
Because of hypertension caused by Cav1.3 gene mutation, a new accurate (targeted) hypertension treatment drug can be developed.
The present invention will be described in detail below with reference to the accompanying drawings and examples.
In order to realize the influence of human gene mutation on the occurrence and development of hypertension and explore a new target point of hypertension treatment, the invention provides a method for discovering a new nosable gene of a hypertension patient through gene sequencing and constructing a human genetic hypertension animal model by using a CRISPR-Cas9 gene editing technology. Animals in the present invention include, but are not limited to: closely related animals such as mice, rats, rabbits, etc., which are used in scientific experiments and in human life (hereinafter, rats are used as an example).
First, animal preparation
The clean wild SD rat and CACNA1D gene point mutation rat are 24 (half male and half female), and the age of the rat is 4 weeks. Free diet, drinking water and life rhythm according to the circadian rhythm.
Animal grouping and model construction
The rat CACNA1D gene (GenBank access number: NM-017298.1; Ensembl: ENSRNOG 00000013147) is located on rat chromosome 16. Contains 51 exons, wherein the ATG initiation codon is located in the 1 st exon, and the TAG termination codon is located in the 51 st exon. And selecting the exon where the site screened in the early stage of the subject group is as the target site. gRNA targeting vectors and donor oligonucleotides (with targeting sequences flanked by 130 bp homologous sequences) were designed: the site of mutation in the donor oligonucleotide will be introduced into this exon by homology directed repair. Silent mutations (from GAG to GAA or from GAC to GAT) will also be introduced to prevent homology directed repair of gRNA binding and re-cleavage of sequences. Cas9 mRNA, gRNA produced by in vitro transcription, and donor oligonucleotide were co-injected into fertilized eggs to breed knock-in rats. These pups will be subjected to sequence analysis.
Grnas of the rat CACNA1D gene, donor oligonucleotides containing mutation sites and silent mutation (GAG to GAA) sites, and Cas9 mRNA were co-injected into fertilized rat eggs to generate targeted knock-in offspring. F0 rats were subjected to sequence analysis and subsequently mated with wild type SD rats to produce F1 generation rats.
Mating the F1 generations to obtain F2 generations, and then dividing the generations into a wild type group, a heterozygous subgroup and a homozygous subgroup according to the difference of genotypes, wherein the specific grouping and modeling methods are as follows:
| wild type group | Heterozygote group | Group of homozygotes | |
| Treatment of | Keeping the room temperature at 22 + -2 deg.C for 12h day and night period, and allowing the solution to be freely used Food and water intake | Keeping room temperature at 22 + -2 deg.C for 12h day and night period, and collecting freely Food and water drinking device | Keeping room temperature at 22 + -2 deg.C for 12h day and night period, and collecting freely Food and water drinking device |
| Time length (moon) | 8 | 8 | 8 |
And (3) measuring physiological and biochemical indexes: blood pressure was measured weekly from 50 days old, after 8 months of feeding, venous blood, content of endothelin-1 in serum was taken from each group of rats after fasting for 12 hours, heart and aorta were collected after sacrifice, and morphology of heart, kidney and aorta was examined.
The model is verified that SBP is larger than or equal to 140mmHg and is used as a standard for successful modeling of the hypertensive WKH rat.
Claims (10)
1. A method for constructing a WKH rat model is characterized by comprising the following steps: adopting gene sequencing technology to detect coding regions and flanking regions of 43 pathogenic genes related to single-gene hypertension of clinical teenager hypertension patients with familial aggregation, predicting the influence of newly-discovered mutant genes on protein structure and function through a plurality of software, and determining the pathogenic genes through pathogenicity judgment; then, the WKH rat model is constructed by utilizing the gene engineering CRISPR/Cas9 technology, the postnatal blood pressure of the mutant gene animal is continuously increased along with the age of the animal, and the systolic pressure of most offspring tail animals is more than 140 mmHg.
2. The method of constructing a WKH rat model according to claim 1, wherein the mutant gene is a gene detection result of a patient with a special type of hypertension.
3. The method for constructing WKH rat model according to claim 1 or 2, wherein said experimental animal is selected from mouse, rat or rabbit.
4. The WKH rat model construction method as claimed in claim 1 or 2, characterized in that the specific gene point mutation F0 mouse is constructed by CRISPR/Cas9 technology.
5. The WKH rat model construction method as claimed in claim 3, characterized in that the specific gene point mutation F0 mouse is constructed by CRISPR/Cas9 technology.
6. The method for constructing WKH rat model according to claim 1, 2 or 5, wherein said specific gene-mutated mouse is the offspring bred from the constructed F0 mouse.
7. The method for constructing WKH rat model according to claim 3, wherein said specific gene-mutated mouse is the offspring of the bred F0 mouse.
8. The method for constructing WKH rat model according to claim 4, wherein said specific gene-mutated mouse is the offspring of the bred F0 mouse.
9. The WKH rat model construction method according to claim 1, 2, 5, 7 or 8, characterized in that the breeding conditions of the mutant rats are SPF-class animal breeding chambers, the temperature is kept at 22 + -2 ℃, the day and night period is 12h, and the mutant rats are fed with food and water freely.
10. The WKH rat model construction method according to claim 3, 4 or 6, characterized in that the breeding conditions of the mutant rats are SPF animal breeding chamber, the room temperature is kept at 22 + -2 ℃, the day and night period is 12h, and the rats can freely take food and drink water.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202011326345.8A CN112522312B (en) | 2020-11-23 | 2020-11-23 | WKH rat model construction method |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202011326345.8A CN112522312B (en) | 2020-11-23 | 2020-11-23 | WKH rat model construction method |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN112522312A true CN112522312A (en) | 2021-03-19 |
| CN112522312B CN112522312B (en) | 2022-10-04 |
Family
ID=74993070
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202011326345.8A Active CN112522312B (en) | 2020-11-23 | 2020-11-23 | WKH rat model construction method |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN112522312B (en) |
Citations (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20070292412A1 (en) * | 2006-05-09 | 2007-12-20 | Oy Jurilab Ltd | Novel genes and markers in type 2 diabetes and obesity |
| WO2014172470A2 (en) * | 2013-04-16 | 2014-10-23 | Whitehead Institute For Biomedical Research | Methods of mutating, modifying or modulating nucleic acid in a cell or nonhuman mammal |
| CN104293831A (en) * | 2014-09-28 | 2015-01-21 | 上海云舜生物技术有限公司 | Method for establishing hypertension mouse model and application of hypertension mouse model |
| CN106929533A (en) * | 2017-03-10 | 2017-07-07 | 上海交通大学医学院附属新华医院 | The construction method of KARS point mutation mouse models and its application |
| CN107475300A (en) * | 2017-09-18 | 2017-12-15 | 上海市同济医院 | The construction method of Ifit3 eKO1 knock out mice animal models and application |
| CN109662974A (en) * | 2018-11-01 | 2019-04-23 | 广西国际壮医医院 | A kind of method for building up of novel rat hypertension model and its application |
| CN110885858A (en) * | 2019-11-01 | 2020-03-17 | 上海交通大学 | Construction method and application of Amy1 gene knockout mouse animal model |
| CN111100877A (en) * | 2018-10-25 | 2020-05-05 | 北京市心肺血管疾病研究所 | Preparation method and application of hypertrophic cardiomyopathy mouse model |
| CN111109199A (en) * | 2019-11-22 | 2020-05-08 | 温州医科大学附属第二医院、温州医科大学附属育英儿童医院 | Slc12a9 gene knockout mouse model and establishment method and application thereof |
-
2020
- 2020-11-23 CN CN202011326345.8A patent/CN112522312B/en active Active
Patent Citations (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20070292412A1 (en) * | 2006-05-09 | 2007-12-20 | Oy Jurilab Ltd | Novel genes and markers in type 2 diabetes and obesity |
| WO2014172470A2 (en) * | 2013-04-16 | 2014-10-23 | Whitehead Institute For Biomedical Research | Methods of mutating, modifying or modulating nucleic acid in a cell or nonhuman mammal |
| CN104293831A (en) * | 2014-09-28 | 2015-01-21 | 上海云舜生物技术有限公司 | Method for establishing hypertension mouse model and application of hypertension mouse model |
| CN106929533A (en) * | 2017-03-10 | 2017-07-07 | 上海交通大学医学院附属新华医院 | The construction method of KARS point mutation mouse models and its application |
| CN107475300A (en) * | 2017-09-18 | 2017-12-15 | 上海市同济医院 | The construction method of Ifit3 eKO1 knock out mice animal models and application |
| CN111100877A (en) * | 2018-10-25 | 2020-05-05 | 北京市心肺血管疾病研究所 | Preparation method and application of hypertrophic cardiomyopathy mouse model |
| CN109662974A (en) * | 2018-11-01 | 2019-04-23 | 广西国际壮医医院 | A kind of method for building up of novel rat hypertension model and its application |
| CN110885858A (en) * | 2019-11-01 | 2020-03-17 | 上海交通大学 | Construction method and application of Amy1 gene knockout mouse animal model |
| CN111109199A (en) * | 2019-11-22 | 2020-05-08 | 温州医科大学附属第二医院、温州医科大学附属育英儿童医院 | Slc12a9 gene knockout mouse model and establishment method and application thereof |
Non-Patent Citations (2)
| Title |
|---|
| YI-DE YANG ET AL.: "Combined effects of the rs9810888 polymorphism in calcium voltage-gated channel subunit alpha1 D（CACNA1D）and lifestyle behaviors on blood pressure level among Chinese children", 《PLOS ONE》 * |
| 陈超: "《新技术与精准医学》", 30 December 2017 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN112522312B (en) | 2022-10-04 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| US20190343094A1 (en) | Method for Constructing PD-1 Gene Modified Humanized Animal Model and Use Thereof | |
| CN109628454B (en) | Construction method of zebra fish glycogen storage disease gys1 and gys2 gene mutant | |
| CN104593418A (en) | Method for establishing humanized rat drug evaluation animal model | |
| WO2018045727A1 (en) | Method for constructing an animal model for mucopolysaccharidosis type ii, and applications thereof | |
| CN112029765A (en) | Method for making Metrnl gene conditional knockout mouse model | |
| KR20120102568A (en) | Tomato plants resulting from the introgression of a trait from solanum pennellii into s. lycopersicum and having an increased yield | |
| CN108753837B (en) | Construction method of rabbit model with hyperlipidemia or atherosclerosis and sgRNA | |
| CN116218905B (en) | Construction and application of point mutation rat epilepsy model | |
| Shi et al. | Modeling human point mutation diseases in Xenopus tropicalis with a modified CRISPR/Cas9 system | |
| CN111471718A (en) | Construction method of zebra fish animal model for screening cardiovascular disease drugs | |
| CN105950654A (en) | A humanized transgenic animal | |
| CN108271740B (en) | Method for establishing neutrophilic granulocyte-deficient atherosclerosis model mouse | |
| Kapuscinski et al. | Nerve growth factor gene and hypertension in spontaneously hypertensive rats | |
| CN108300738B (en) | Preparation method of humanized mouse model with NOD genetic background and neutrophilic granulocyte deletion | |
| CN112522312B (en) | WKH rat model construction method | |
| WO2024103631A1 (en) | Transgenic mouse with ggc repeat expansion mutation in notch2nlc gene, construction method therefor, and use thereof | |
| JP2005530509A (en) | Methods for the development of animal models | |
| CN108018315A (en) | A kind of application of separated gene order in the blue or green Medaka albefaction strain of Japan is prepared | |
| CN106399369B (en) | Build the method and targeting vector and kit in the mouse model of hippocampus regiospecificity knockout IKK α genes | |
| CN112980881B (en) | Construction method and application of Arvcf gene knockout animal model | |
| Zhang et al. | A single silk‐and multiple pollen‐expressed PMEs at the Ga1 locus modulate maize unilateral cross‐incompatibility | |
| CN114457114A (en) | Construction method of Fars2 gene conditional knockout animal model | |
| CA2616258C (en) | Non-human animal model for cardiovascular disease characterized by a disrupted fibulin-4 gene | |
| CN112626122A (en) | hKDR humanized mouse model and establishing method and application thereof | |
| Chai et al. | The gap in research on polyploidization between plants and vertebrates: model systems and strategic challenges |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |