CN108271740B - Method for establishing neutrophilic granulocyte-deficient atherosclerosis model mouse - Google Patents
Method for establishing neutrophilic granulocyte-deficient atherosclerosis model mouse Download PDFInfo
- Publication number
- CN108271740B CN108271740B CN201810078748.1A CN201810078748A CN108271740B CN 108271740 B CN108271740 B CN 108271740B CN 201810078748 A CN201810078748 A CN 201810078748A CN 108271740 B CN108271740 B CN 108271740B
- Authority
- CN
- China
- Prior art keywords
- mouse
- gfi1
- atherosclerosis
- apoe
- mice
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
- 201000001320 Atherosclerosis Diseases 0.000 title claims abstract description 73
- 210000003714 granulocyte Anatomy 0.000 title claims abstract description 34
- 238000000034 method Methods 0.000 title claims abstract description 34
- 230000003448 neutrophilic effect Effects 0.000 title claims abstract description 30
- 230000002950 deficient Effects 0.000 title claims abstract description 24
- 101150023475 Gfi1 gene Proteins 0.000 claims abstract description 42
- 230000035772 mutation Effects 0.000 claims abstract description 35
- 230000007812 deficiency Effects 0.000 claims abstract description 11
- 230000015572 biosynthetic process Effects 0.000 claims abstract description 8
- 230000008439 repair process Effects 0.000 claims abstract description 7
- 241000699666 Mus <mouse, genus> Species 0.000 claims description 80
- 241000699670 Mus sp. Species 0.000 claims description 43
- 210000000440 neutrophil Anatomy 0.000 claims description 28
- 101150037123 APOE gene Proteins 0.000 claims description 23
- 108090000623 proteins and genes Proteins 0.000 claims description 19
- 238000003209 gene knockout Methods 0.000 claims description 16
- 238000009395 breeding Methods 0.000 claims description 11
- 230000001488 breeding effect Effects 0.000 claims description 10
- 238000000684 flow cytometry Methods 0.000 claims description 5
- 230000008569 process Effects 0.000 claims description 5
- 230000000694 effects Effects 0.000 claims description 4
- 101001046686 Homo sapiens Integrin alpha-M Proteins 0.000 claims description 3
- 102100022338 Integrin alpha-M Human genes 0.000 claims description 3
- 238000003205 genotyping method Methods 0.000 claims description 3
- 238000007480 sanger sequencing Methods 0.000 claims description 3
- 102100024222 B-lymphocyte antigen CD19 Human genes 0.000 claims description 2
- 102100032912 CD44 antigen Human genes 0.000 claims description 2
- 101000980825 Homo sapiens B-lymphocyte antigen CD19 Proteins 0.000 claims description 2
- 101000868273 Homo sapiens CD44 antigen Proteins 0.000 claims description 2
- 101100341519 Homo sapiens ITGAX gene Proteins 0.000 claims description 2
- 101001057504 Homo sapiens Interferon-stimulated gene 20 kDa protein Proteins 0.000 claims description 2
- 101001055144 Homo sapiens Interleukin-2 receptor subunit alpha Proteins 0.000 claims description 2
- 101000738771 Homo sapiens Receptor-type tyrosine-protein phosphatase C Proteins 0.000 claims description 2
- 101000716102 Homo sapiens T-cell surface glycoprotein CD4 Proteins 0.000 claims description 2
- 101000934341 Homo sapiens T-cell surface glycoprotein CD5 Proteins 0.000 claims description 2
- 101000946843 Homo sapiens T-cell surface glycoprotein CD8 alpha chain Proteins 0.000 claims description 2
- 102100022297 Integrin alpha-X Human genes 0.000 claims description 2
- 102100027268 Interferon-stimulated gene 20 kDa protein Human genes 0.000 claims description 2
- 102100037422 Receptor-type tyrosine-protein phosphatase C Human genes 0.000 claims description 2
- 102100036011 T-cell surface glycoprotein CD4 Human genes 0.000 claims description 2
- 102100025244 T-cell surface glycoprotein CD5 Human genes 0.000 claims description 2
- 102100034922 T-cell surface glycoprotein CD8 alpha chain Human genes 0.000 claims description 2
- 238000001502 gel electrophoresis Methods 0.000 claims description 2
- 230000003993 interaction Effects 0.000 claims description 2
- 102000013918 Apolipoproteins E Human genes 0.000 claims 1
- 108010025628 Apolipoproteins E Proteins 0.000 claims 1
- -1 LY6G Proteins 0.000 claims 1
- 230000003143 atherosclerotic effect Effects 0.000 abstract description 6
- 238000002474 experimental method Methods 0.000 abstract description 6
- 238000013258 ApoE Receptor knockout mouse model Methods 0.000 abstract description 4
- 238000003745 diagnosis Methods 0.000 abstract description 4
- 208000024891 symptom Diseases 0.000 abstract description 4
- 208000037260 Atherosclerotic Plaque Diseases 0.000 abstract description 3
- 230000002068 genetic effect Effects 0.000 abstract description 3
- 238000012239 gene modification Methods 0.000 abstract description 2
- 238000010353 genetic engineering Methods 0.000 abstract description 2
- 230000005017 genetic modification Effects 0.000 abstract description 2
- 235000013617 genetically modified food Nutrition 0.000 abstract description 2
- 230000036285 pathological change Effects 0.000 abstract 1
- 231100000915 pathological change Toxicity 0.000 abstract 1
- 238000010172 mouse model Methods 0.000 description 11
- NPGIHFRTRXVWOY-UHFFFAOYSA-N Oil red O Chemical compound Cc1ccc(C)c(c1)N=Nc1cc(C)c(cc1C)N=Nc1c(O)ccc2ccccc12 NPGIHFRTRXVWOY-UHFFFAOYSA-N 0.000 description 10
- 239000000047 product Substances 0.000 description 9
- 235000009200 high fat diet Nutrition 0.000 description 8
- 239000002773 nucleotide Substances 0.000 description 8
- 125000003729 nucleotide group Chemical group 0.000 description 8
- 238000010186 staining Methods 0.000 description 8
- 230000003321 amplification Effects 0.000 description 7
- 238000003199 nucleic acid amplification method Methods 0.000 description 7
- 238000001514 detection method Methods 0.000 description 6
- 238000011534 incubation Methods 0.000 description 6
- 239000000203 mixture Substances 0.000 description 6
- 239000012634 fragment Substances 0.000 description 5
- 210000002966 serum Anatomy 0.000 description 5
- 206010061598 Immunodeficiency Diseases 0.000 description 4
- 208000029462 Immunodeficiency disease Diseases 0.000 description 4
- 238000010171 animal model Methods 0.000 description 4
- 238000002156 mixing Methods 0.000 description 4
- 238000011160 research Methods 0.000 description 4
- 210000004369 blood Anatomy 0.000 description 3
- 239000008280 blood Substances 0.000 description 3
- 208000026106 cerebrovascular disease Diseases 0.000 description 3
- 238000012224 gene deletion Methods 0.000 description 3
- 230000007813 immunodeficiency Effects 0.000 description 3
- 238000007619 statistical method Methods 0.000 description 3
- 238000012360 testing method Methods 0.000 description 3
- 201000010000 Agranulocytosis Diseases 0.000 description 2
- 208000024172 Cardiovascular disease Diseases 0.000 description 2
- 208000014882 Carotid artery disease Diseases 0.000 description 2
- 208000007530 Essential hypertension Diseases 0.000 description 2
- 206010018687 Granulocytopenia Diseases 0.000 description 2
- 108010010234 HDL Lipoproteins Proteins 0.000 description 2
- 102000015779 HDL Lipoproteins Human genes 0.000 description 2
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 description 2
- 108010007622 LDL Lipoproteins Proteins 0.000 description 2
- 102000007330 LDL Lipoproteins Human genes 0.000 description 2
- 229930040373 Paraformaldehyde Natural products 0.000 description 2
- 208000037876 carotid Atherosclerosis Diseases 0.000 description 2
- 210000004027 cell Anatomy 0.000 description 2
- HVYWMOMLDIMFJA-DPAQBDIFSA-N cholesterol Chemical compound C1C=C2C[C@@H](O)CC[C@]2(C)[C@@H]2[C@@H]1[C@@H]1CC[C@H]([C@H](C)CCCC(C)C)[C@@]1(C)CC2 HVYWMOMLDIMFJA-DPAQBDIFSA-N 0.000 description 2
- 238000010276 construction Methods 0.000 description 2
- 238000012217 deletion Methods 0.000 description 2
- 230000037430 deletion Effects 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- 238000010586 diagram Methods 0.000 description 2
- 201000010099 disease Diseases 0.000 description 2
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 2
- 239000012154 double-distilled water Substances 0.000 description 2
- 230000002526 effect on cardiovascular system Effects 0.000 description 2
- 210000003743 erythrocyte Anatomy 0.000 description 2
- 230000036541 health Effects 0.000 description 2
- 238000009396 hybridization Methods 0.000 description 2
- 210000000987 immune system Anatomy 0.000 description 2
- 230000003902 lesion Effects 0.000 description 2
- 210000004698 lymphocyte Anatomy 0.000 description 2
- 239000006166 lysate Substances 0.000 description 2
- 238000005259 measurement Methods 0.000 description 2
- 229920002866 paraformaldehyde Polymers 0.000 description 2
- 230000008506 pathogenesis Effects 0.000 description 2
- 239000011550 stock solution Substances 0.000 description 2
- 230000000153 supplemental effect Effects 0.000 description 2
- UFTFJSFQGQCHQW-UHFFFAOYSA-N triformin Chemical compound O=COCC(OC=O)COC=O UFTFJSFQGQCHQW-UHFFFAOYSA-N 0.000 description 2
- 239000012224 working solution Substances 0.000 description 2
- 206010061218 Inflammation Diseases 0.000 description 1
- 101100055876 Mus musculus Apoe gene Proteins 0.000 description 1
- 208000018262 Peripheral vascular disease Diseases 0.000 description 1
- 238000008050 Total Bilirubin Reagent Methods 0.000 description 1
- 206010072810 Vascular wall hypertrophy Diseases 0.000 description 1
- 238000000246 agarose gel electrophoresis Methods 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 230000000844 anti-bacterial effect Effects 0.000 description 1
- 210000000709 aorta Anatomy 0.000 description 1
- 210000001367 artery Anatomy 0.000 description 1
- 230000036523 atherogenesis Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000003115 biocidal effect Effects 0.000 description 1
- 210000000601 blood cell Anatomy 0.000 description 1
- 206010008118 cerebral infarction Diseases 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 230000003399 chemotactic effect Effects 0.000 description 1
- 208000037976 chronic inflammation Diseases 0.000 description 1
- 208000037893 chronic inflammatory disorder Diseases 0.000 description 1
- 208000029078 coronary artery disease Diseases 0.000 description 1
- 230000006378 damage Effects 0.000 description 1
- 235000005911 diet Nutrition 0.000 description 1
- 230000037213 diet Effects 0.000 description 1
- 238000007865 diluting Methods 0.000 description 1
- 239000012153 distilled water Substances 0.000 description 1
- 238000000556 factor analysis Methods 0.000 description 1
- 230000035558 fertility Effects 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 230000036449 good health Effects 0.000 description 1
- 210000005003 heart tissue Anatomy 0.000 description 1
- 238000013394 immunophenotyping Methods 0.000 description 1
- 230000004054 inflammatory process Effects 0.000 description 1
- 230000028709 inflammatory response Effects 0.000 description 1
- 230000007774 longterm Effects 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 230000001404 mediated effect Effects 0.000 description 1
- 230000000242 pagocytic effect Effects 0.000 description 1
- 210000004976 peripheral blood cell Anatomy 0.000 description 1
- 239000002244 precipitate Substances 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 238000012163 sequencing technique Methods 0.000 description 1
- 230000004083 survival effect Effects 0.000 description 1
- 210000001519 tissue Anatomy 0.000 description 1
- 238000012795 verification Methods 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
- 230000003442 weekly effect Effects 0.000 description 1
Images
Classifications
-
- 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
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
-
- 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
- A01K2207/12—Animals modified by administration of exogenous cells
-
- 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
- A01K2217/07—Animals genetically altered by homologous recombination
- A01K2217/075—Animals genetically altered by homologous recombination inducing loss of function, i.e. knock out
-
- 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
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/156—Polymorphic or mutational markers
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Analytical Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Health & Medical Sciences (AREA)
- Zoology (AREA)
- Organic Chemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Biotechnology (AREA)
- General Health & Medical Sciences (AREA)
- Wood Science & Technology (AREA)
- Environmental Sciences (AREA)
- Animal Behavior & Ethology (AREA)
- Animal Husbandry (AREA)
- Physics & Mathematics (AREA)
- Biophysics (AREA)
- Immunology (AREA)
- Microbiology (AREA)
- Molecular Biology (AREA)
- Biodiversity & Conservation Biology (AREA)
- Veterinary Medicine (AREA)
- Biochemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- Genetics & Genomics (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention relates to a method for establishing a neutrophile granulocyte-deleted atherosclerosis model mouse, belonging to the technical field of genetic engineering and genetic modification. According to the invention, Gfi1 point mutation is introduced into ApoE knockout mouse genome for the first time, and the first neutrophilic granulocyte deficiency atherosclerosis mouse genetic model is constructed. Experiments prove that in the model mouse, atherosclerotic plaques are obviously reduced, and the atherosclerotic symptoms are obviously relieved, which shows that the neutrophile granulocyte-deficient atherosclerosis model mouse is successfully constructed, and the neutrophile granulocyte deficiency can relieve the pathological change degree of atherosclerosis. The model mouse with neutrophilic granulocyte deficiency and atherosclerosis obtained by the invention has very important potential application value for uncovering the role of neutrophilic granulocyte in formation and repair of atherosclerosis and diagnosis and treatment of atherosclerosis.
Description
Technical Field
The invention relates to a method for establishing a neutrophile granulocyte-deleted atherosclerosis model mouse, belonging to the technical field of genetic engineering and genetic modification.
Background
Cardiovascular and cerebrovascular diseases are difficult to cure and high in death rate, and become the disease causing the most death worldwide, atherosclerosis is one of the main causes of cardiovascular and cerebrovascular diseases, the pathogenesis of the atherosclerosis is complex, the degree of harm to health is serious, and the mechanism of the atherosclerosis needs to be deeply researched. Atherosclerosis (AS) is a major cause of coronary heart disease, cerebral infarction, and peripheral vascular disease, but there is no suitable animal model for studying its pathogenesis and clinical treatment.
Neutrophils (neutrophiles) are one of the blood cell components in the blood, and have chemotactic, phagocytic and bactericidal effects. Atherosclerosis is a chronic inflammatory disease that severely threatens human health, while neutrophils are the main cell population for the inflammatory response. The relation between the carotid atherosclerosis of the essential hypertension patients and the ratio of the neutrophils to the lymphocytes and the total bilirubin level of the serum, the higher the index bleed atherosclerosis in southern medical university, Liudongfeng, etc., the higher the patient's neutrophil/lymphocyte ratio NLR, and the result is also confirmed by multi-factor analysis, wherein the NLR is an independent risk factor of the carotid atherosclerosis of the essential hypertension patients, and the suggestion that the inflammation mediated by peripheral blood cells plays an important role in the reaction of intimal thickening of arteries.
However, the role of neutrophils in atherogenesis has been overlooked due to the lack of a suitable animal model that can be used to study the association between atherosclerosis and neutrophils. Research on atherosclerosis requires long-term follow-up of animal models, whereas currently existing neutrophilic granulocytopenia mice survive only for weeks and require antibiotic feeding in an SPF environment. At present, no suitable animal model can be used when researching the problems of whether neutrophils are needed in the process of atherosclerosis formation, whether the neutrophils participate in the repair of atherosclerosis and the like.
Disclosure of Invention
The invention aims to provide a method for establishing a neutrophil-deficient atherosclerosis model mouse, which is simple and rapid, and the obtained model mouse can be conveniently used for researching the effect of neutrophil on atherosclerosis.
In order to achieve the purpose, the invention adopts the technical scheme that:
a method for establishing a neutrophil-deficient atherosclerosis model mouse comprises the following steps:
1) hybridizing an Gfi1 point mutation mouse with an ApoE gene knockout mouse to obtain an F1 generation mouse with heterozygote of double genes;
2) selfing an F1 generation mouse with heterozygote double genes to obtain an F2 generation mouse, and carrying out genotype identification on the F2 generation mouse; selecting male mice of which both Gfi1 point mutation and ApoE gene knockout are homozygote, female mice of which Gfi1 point mutation is heterozygote and ApoE gene knockout is homozygote from F2 generation mice;
3) and hybridizing the male mouse and the female mouse, wherein Gfi1 point mutation and ApoE gene knockout in offspring are homozygote mice, namely the model mouse of neutrophilic granulocyte deletion atherosclerosis.
The Gfi1 point mutation mouse selected in the invention is a neutrophil-deleted mouse model which can survive for a long time, and in order to determine whether neutrophil is needed in the process of atherosclerosis formation and whether neutrophil participates in the repair of atherosclerosis, Gfi1 point mutation is introduced into ApoE knockout mouse genome for the first time, and the first neutrophil-deleted atherosclerosis mouse genetic model is constructed. According to the breeding method, a female mouse with Gfi1 point mutation as a heterozygote and ApoE gene knockout as a homozygote is selected for breeding, the immune system of the female mouse is consistent with the wild type, the female mouse is completely normal in performance and can be normally bred and suckled, and therefore it is guaranteed that a sufficient number of model mice with both Gfi1 point mutation and ApoE gene knockout as homozygotes can be obtained in subsequent experiments.
The Gfi1 genotype of the F2 mouse is identified by the following primers in the step 2):
gfi 1-f: GGAGCTACGCTTTTGTCCTG, Gfi 1-r: CCTGTGTGGATGAAGGTGTG are provided. The nucleotide sequence of the normal Gfi1 gene fragment amplified by the pair of primers is shown as SEQ ID NO.3, and the 288 th site of the nucleotide sequence of the point mutation Gfi1 gene fragment is mutated from G to A. Specifically, the point mutation of the Gfi1 gene can be detected by Sanger sequencing, and the Gfi1 genotype of the mouse can be conveniently and quickly detected by using the method. If the 288 th nucleotide is only G, the gene is a normal gene; if the 288 th nucleotide is only A, the mutant is a point mutation homozygote; and the 288 th nucleotide is G/A, and is heterozygote for point mutation.
Identifying the ApoE genotype of the F2 mouse by adopting the following primers in the step 2):
ApoE-1：GCCTAGCCGAGGGAGAGCCG，
ApoE-2：TGTGACTTGGGAGCTCTGCAGC，
ApoE-3: GCCGCCCCGACTGCATCT, which are supplied by the Jackson laboratory when purchasing ApoE mice.
Wherein, ApoE-1 is a common primer, wherein the amplified fragments of ApoE-1 and ApoE-2 are shown in SEQ ID NO.7, and the total length is 155 bp; the fragments amplified by ApoE-1 and ApoE-3 are shown in SEQ ID NO.8 as 245bp in total length. Detection of ApoE genes can be performed using gel electrophoresis. If the amplification product is only a band of 155bp, the gene is a normal gene; if the amplification product is only one band of 245bp, the gene is knocked out and homozygote exists; if the amplification product has two bands, one is 155bp, and the other is 245bp, the gene is a knockout heterozygote.
Specifically, in step 2), after genotyping F2 generation mice, immunophenotype analysis was performed on F2 generation mice using flow cytometry. The antibodies are CD4, CD5, CD8, CD11B, CD11C, CD19, CD25, CD44, CD45, LY6G, and NK 1.1. More specifically, firstly preparing an antibody mixture, then sucking 40 mul of fresh blood, adding 10 mul of the antibody mixture, fully shaking and uniformly mixing, placing in a refrigerator at 4 ℃ for dark incubation for 30min, after the incubation is finished, adding 400 mul of erythrocyte lysate, fully shaking and uniformly mixing, placing at room temperature for dark incubation for 10min, after the reaction is finished, and then detecting by adopting a flow cytometer. By adopting the method, whether the neutrophils in the F2 generation mouse are deleted can be further determined, and a mouse individual with completely deleted neutrophils can be further screened.
In the breeding process of the model mouse, a male mouse with Gfi1 point mutation and ApoE gene knockout both homozygote, a female mouse with Gfi1 point mutation as heterozygote and ApoE gene knockout as homozygote are selected to breed offspring. The immune system of the female mouse is completely normal, and breeding is not influenced, so that enough offspring mice can be obtained, and up to 50% of the offspring mice are homozygotes, thereby ensuring the smooth development of subsequent experiments.
The present invention will result in mouse neutrophilic granulocyte deficiency but normal survival mouse mutation site Gfi1 for the first timeC318YMouse model of atherosclerosis with genetic background introduced by hybridization of ApoE gene deletion (ApoE)-/-) Through hybridization and screening, the homozygote mouse ApoE with double gene deletion is obtained-/-Gfi1C318Y/C318YIt was experimentally confirmed that in ApoE-/-Gfi1C318Y/C318YIn mice, the atherosclerosis symptom is obviously relieved, which shows that the neutrophilic granulocyte-deficient atherosclerosis model mouse is successfully constructed.
The neutrophil-loss atherosclerosis model mouse obtained by the establishing method is applied to research of interaction between neutrophil loss and atherosclerosis. In particular, the application of the neutrophile granulocyte deficiency atherosclerosis model mouse in researching the effect of neutrophile granulocytes on the formation and repair of atherosclerosis. Or the application of the neutrophile granulocyte deficiency atherosclerosis model mouse in researching a diagnosis and treatment method of atherosclerosis.
The obtained model mouse with neutrophilic granulocyte deficiency and atherosclerosis is further subjected to experimental study, and the result shows that the neutrophilic granulocyte deficiency can obviously reduce the formation of atherosclerotic plaques. The model mouse with neutrophilic granulocyte deficiency and atherosclerosis obtained by the invention has very important potential application value for uncovering the role of neutrophilic granulocyte in formation and repair of atherosclerosis and diagnosis and treatment of atherosclerosis.
The invention has the following beneficial effects:
1. obtaining an atherosclerosis model mouse which is deficient in neutrophils and can normally survive for the first time;
2. it is proposed for the first time to use as male parent (ApoE genotype) male mice which are immunodeficient but can normally breed in breeding mice with severe immunodeficiency-/-Gfi1C318Y/C318Y) Heterozygote mice with normal immunophenotype are used as female parents (ApoE genotype)-/-Gfi1+/C318Y) And hybridizing to obtain enough homozygous immune-deficient mice (ApoE) while ensuring smooth breeding-/-Gfi1C318Y/C318Y) The method is used for subsequent experiments, and provides a new idea for breeding mice with similar immunodeficiency;
3. the relation between the neutrophils and the atherosclerosis is determined for the first time, namely the neutrophilic granulocyte deletion can obviously relieve the symptoms of the atherosclerotic disease, which opens a new direction for deeply developing the research of the atherosclerosis, provides more choices for the clinical treatment of the atherosclerosis and has very important theoretical and practical significance.
Drawings
FIG. 1 is a graph showing the sequencing verification result of Gfi1 point mutation site of F2 mouse;
FIG. 2 is a diagram showing the result of ApoE genotype determination in mouse generation F2;
FIG. 3 is a graph of immunophenotypic results for a sex granulocyte deficient atherosclerotic model mouse;
FIG. 4 is a graph showing the results of serum biochemical indicator measurements after the model and control mice were fed high-fat diet for 12 weeks;
FIG. 5 is a graph showing the results of oil red O and H & E staining of heart cryosections after the model and control mice were fed with high-fat diet for 12 weeks;
FIG. 6 is a graph showing the results of aortic oil red O staining after the model and control mice were fed with high-fat diet for 12 weeks.
Detailed Description
The present invention will be described in further detail with reference to specific examples. Gfi1 point mutation mouse (genotype Gfi 1) in the following examples and experimental examplesC318Y/C318Y) Supplied by Bernard Malissen laboratory, France; ApoE knockout mice (genotype is ApoE)-/-) Purchased by Jackson Laboratory, usa. The formula of the high-fat feed is referred to the Research Diets company No. D12108C. Preparation of oil red O stock solution: 100% isopropanol 100mL +0.5g oil red O, storing in a 4 degree refrigerator, preparing oil red O working solution (stock solution and distilled water are diluted according to a ratio of 3: 2) when in use, diluting and filtering before use, wherein the oil red O working solution should be wine red and have no precipitate). Other experimental articles are not described and are all commercial products.
Example 1
The method for establishing the neutrophil-deficient atherosclerosis model mouse comprises the following steps:
1) gfi1 point mutation mouse (genotype Gfi 1)C318Y/C318Y) With ApoE knockout mice (genotype is ApoE)-/-) Hybridizing to obtain F1 generation mouse, wherein the double gene sites of the F1 generation mouse are heterozygote (the genotype is ApoE)+/-Gfi1+/C318Y)。
2) Mice with heterozygote double gene loci of F1 generation are selfed to obtain mice of F2 generation.
A. By using sanger sequencing, mice with Gfi1 point mutation sites as heterozygotes and homozygotes can be selected, and the detection primer sequence is as follows: gfi 1-f: GGAGCTACGCTTTTGTCCTG (shown as SEQ ID NO. 1), Gfi 1-r: CCTGTGTGGATGAAGGTGTG (shown as SEQ ID NO. 2), and the size of the amplified product is 429 bp.
The PCR system is as follows: (Vazyme, P111)2 Taq Master PCR MIX: 10 μ L of each, Gfi1-F (5 μ M) and Gfi1-R (5 μ M), 0.5 μ L; genomic DNA: 10 ng; supplemental ddH2O to the total volume of 20 mu L; the procedure is as follows: 95 ℃ for 5 min; 95 ℃, 30s, 60 ℃, 30s, 72 ℃, 40 s; 72 ℃ for 10 min; the number of PCR cycles was 35.
The detection result is shown in SEQ ID NO.3, wherein the 288 th nucleotide is G only, and the gene is a normal gene (WT); the 288 th nucleotide is only A, and is a point mutation homozygote (Gfi 1)C318Y/C318Y) (ii) a The 288 th nucleotide is G/A, and is a point mutation heterozygote (Gfi 1)+/C318Y)。
B. By using 1.5% agarose gel electrophoresis, a mouse with an ApoE gene locus as a homozygote can be selected, and the detection primer sequence is as follows: ApoE-1: GCC TAG CCG AGG GAG AGC CG (shown in SEQ ID NO. 4), ApoE-2: TGTGAC TTG GGA GCT CTG CAG C (shown in SEQ ID NO. 5), ApoE-3: GCC GCC CCG ACT GCA TCT (shown in SEQ ID NO. 6).
The PCR system is as follows: (Vazyme, P111)2 Taq Master PCR MIX: 10 μ L of each, Gfi1-F (5 μ M) and Gfi1-R (5 μ M), 0.5 μ L; genomic DNA: 10 ng; supplemental ddH2O to the total volume of 20 mu L; the procedure is as follows: 95 ℃ for 5 min; 95 ℃, 30s, 60 ℃, 30s, 72 ℃, 40 s; 72 ℃ for 10 min; the number of PCR cycles was 35.
The detection result is shown in FIG. 2, wherein the amplification product is only a band of 155bp, which is a normal gene (WT); the amplification product is only one band of 245bp, and is a knockout gene homozygote (ApoE)-/-) (ii) a The amplification product has two bands, one is 155bp, the other is 245bp, and the amplification product is a knockout gene heterozygote (ApoE)+/-)。
C. Using flow cytometry, homozygous mice with neutrophilic granulocyte depletion can be further tested and selected using a loss cytometer model number CANTO II (BD, USA) with antibody combinations as shown in Table 1.
The method comprises the following steps: firstly preparing an antibody mixture, then sucking 40 mu l of fresh blood, adding 10 mu l of the antibody mixture, fully shaking and uniformly mixing, placing in a refrigerator at 4 ℃ for dark incubation for 30min, adding 400 mu l of erythrocyte lysate after incubation is finished, fully shaking and uniformly mixing, placing at room temperature for dark incubation for 10min, finishing reaction, and then detecting by adopting a flow cytometer.
Table 1 name and brand of antibody used for flow cytometry
The result of the detection is shown in FIG. 3, and it can be seen that CD11B+LY6G+The positive cells (i.e., neutrophils, marked in the boxes in the figure) had disappeared, indicating that the mature neutrophils in the model mice obtained from the construction had been completely deleted.
3) Because the Gfi1 point mutation site is homozygousMouse (genotype Gfi 1)C318Y/C318Y) Since neutrophilic granulocytes are deleted, serious immunodeficiency is caused, the fecundity is low, and if the neutrophilic granulocytes are used as parents for breeding, enough bigene homozygote mice are difficult to obtain for experiments, so that the invention selects male mice (the genotype is ApoE) with both Gfi1 point mutation and ApoE gene homozygote from F2 generation mice-/-Gfi1C318Y/C318YImmunodeficient but normal breeding), Gfi1 point mutation to heterozygous and ApoE gene homozygote female mouse (ApoE genotype)-/-Gfi1+/C318YNormal neutrophilic granulocyte, no immunodeficiency and good health state) to obtain Gfi1 point mutation and ApoE gene homozygote mouse (ApoE gene type is ApoE)-/-Gfi1C318Y/C318Y) The mouse is a new neutrophilic granulocyte-deficient atherosclerosis mouse model when used for subsequent experiments.
The F2 mice were successfully selected for double gene deletion (genotype is ApoE) by genotyping (FIGS. 1 and 2) and immunophenotyping (FIG. 3)-/-Gfi1C318Y/C318Y) But also neutrophil-depleted progeny mice. In the subsequent breeding process of model mice, male mice with Gfi1 point mutation and ApoE gene knockout homozygote, female mice with Gfi1 point mutation as heterozygote and ApoE gene knockout as homozygote are selected for breeding offspring.
Test example 1
The mouse model of neutrophile-deficient atherosclerosis obtained in example 1 (genotype ApoE)-/-Gfi1C318Y/C318Y) And control mice (genotype ApoE)-/-) And (5) performing high fat feeding.
Model mouse of neutrophilic granulocyte-deficient atherosclerosis (ApoE genotype)-/-Gfi1C318Y/C318Y) And control mice (genotype ApoE)-/-) High fat diet feeding was performed, during which the weight change was monitored weekly, taking care to weigh at the same time point every week. As a result, the weight of the neutrophilic granulocyte-deficient atherosclerosis model mouse is not significantly different from that of the control mouse.
Model mouse of neutrophilic granulocyte-deficient atherosclerosis (ApoE genotype)-/-Gfi1C318Y/C318Y) And control mice (genotype ApoE)-/-) After feeding with high-fat diet for 12 weeks, serum was obtained, biochemical indicators such as serum Total Cholesterol (TC), Triglyceride (TG), high-density lipoprotein (HDL), and low-density lipoprotein (LDL) (ADVIA 2400, SIEMENS were used as a measurement instrument), and statistical analysis was performed on the data.
And (5) counting results: there was no significant difference in serum biochemical indicators in the neutrophil-deficient atherosclerosis model mice compared to the control mice (as shown in fig. 4).
Test example 2
Model mouse of neutrophilic granulocyte-deficient atherosclerosis (ApoE genotype)-/-Gfi1C318Y/C318Y) And control mice (genotype ApoE)-/-) After 12 weeks of high-fat diet feeding, heart tissue was harvested, frozen sections (10um) after fixation (4% paraformaldehyde, overnight fixation) and embedding (embedding using OCT), followed by oil red O staining and H&E, staining, counting the area of the plaque, and carrying out statistical analysis on the data.
Oil red O staining and H&The staining results are shown in FIG. 5-A, in which ApoE in control group is shown in FIG. 5-A-/-Mouse and experimental group ApoE-/-Gfi1C318Y/C318YSchematic representation of a sample of a heart portion. Therefore, the area of the heart plaque of the mice in the experimental group is obviously reduced; statistical results of plaque area As shown in FIG. 5-B, atherosclerosis was induced by feeding high-fat diet, and neutrophile-deficient atherosclerosis model mice (ApoE) were found-/-Gfi1C318Y/C318Y) And atherosclerosis model mice (ApoE)-/-) In contrast, there was a significant reduction in plaque area, and the results indicated that neutrophilic granulocytopenia reduced the extent of atherosclerotic lesions.
The neutrophile granulocyte-deleted atherosclerosis model mouse obtained by the establishing method can be used for researching the effect of neutrophile granulocytes on the formation and repair of atherosclerosis. The neutrophilic granulocyte-deleted atherosclerosis model mouse obtained by the establishing method can also be used for researching a diagnosis and treatment method of atherosclerosis.
Test example 3
Model mouse of neutrophilic granulocyte-deficient atherosclerosis (ApoE genotype)-/-Gfi1C318Y/C318Y) And control mice (genotype ApoE)-/-) After feeding with high-fat feed for 12 weeks, aortic tissues were obtained, and after fixation with 4% paraformaldehyde for development, oil red O staining was performed, plaque area was counted, and statistical analysis was performed on the data.
The oil red O staining results are shown in FIG. 6-A, which is ApoE of control group-/-Mouse and experimental group ApoE-/-Gfi1C318Y/C318YA schematic diagram of a mouse aorta sample, from which it can be seen that the aortic plaque area in the experimental group is significantly reduced; statistical results of plaque area As shown in FIG. 6-B, atherosclerosis was induced by feeding high-fat diet, and as such, neutrophile-deficient atherosclerosis model mice (ApoE) were found-/-Gfi1C318Y/C318Y) The plaque area of (A) is significantly smaller than that of an atherosclerosis model mouse (ApoE)-/-)。
From the above experimental results, it can be seen that, compared with the wild type control, in the mouse of Gfi1 and ApoE double gene homozygote, the atherosclerotic plaque is significantly reduced, and the atherosclerotic symptom is significantly reduced, which indicates that the construction of the neutrophil-deficient atherosclerosis model mouse is successful, and the neutrophil deficiency can reduce the atherosclerotic lesion degree.
<110> New countryside medical college
<120> method for establishing neutrophilic granulocyte-deleted atherosclerosis model mouse
<160>6
<170>SIPOSequenceListing 1.0
<211>20
<212>DNA
<213> Artificial sequence
<221>Gfi1-f
<400>1
<211>20
<212>DNA
<213> Artificial sequence
<221>Gfi1-r
<400>2
<211>429
<212>DNA
<213> mice
<221> Gfi1 Gene fragment
<400>3
ggagctacgc ttttgtcctg gggtgtggag ggttgtggct gtaaagtgtt ttagtagtta 60
atcagtaggt gttgaatgat ggggtggagg gagagaggag agggtttctg gctctgggag 120
atcagtagat gttggagaga gagagagaga gagagagaga gagagagaga gagagagaga 180
gagagagaga gagcacaagc gccaaggagg ctgctgaagg agcggcacat ttcttcctcc 240
tctcttcctc tctgctctgc aggaacgcag ctttgactgt aagatctgtg gcaagagctt 300
caagaggtca tccacgctgt ccacacatct gctcattcac tcggacaccc ggccctatcc 360
ctgtcagtac tgtggcaaaa gattccacca gaagtcagat atgaagaaac acaccttcat 420
ccacacagg 429
<211>20
<212>DNA
<213> Artificial sequence
<221>ApoE-1
<400>4
<211>22
<212>DNA
<213> Artificial sequence
<221>ApoE-2
<400>5
tgtgacttgg gagctctgca gc 22
<211>18
<212>DNA
<213> Artificial sequence
<221>ApoE-3
<400>6
gccgccccga ctgcatct 18
Claims (10)
1. A method for establishing a neutrophil-deficient atherosclerosis model mouse is characterized by comprising the following steps: the method comprises the following steps:
1) hybridizing an Gfi1 point mutation mouse with an ApoE gene knockout mouse to obtain an F1 generation mouse with heterozygote of double genes;
2) selfing an F1 generation mouse with heterozygote double genes to obtain an F2 generation mouse, and carrying out genotype identification on the F2 generation mouse; selecting male mice of which both Gfi1 point mutation and ApoE gene knockout are homozygote, female mice of which Gfi1 point mutation is heterozygote and ApoE gene knockout is homozygote from F2 generation mice;
3) and hybridizing the male mouse and the female mouse, wherein Gfi1 point mutation and ApoE gene knockout in offspring are homozygote mice, namely the neutrophilic granulocyte-deleted atherosclerosis model mouse.
2. The method of establishing according to claim 1, wherein: the Gfi1 genotype of the F2 mouse is identified by the following primers in the step 2):
Gfi1-f：GGAGCTACGCTTTTGTCCTG，Gfi1-r：CCTGTGTGGATGAAGGTGTG。
3. the method of establishing according to claim 1, wherein: identifying the ApoE genotype of the F2 mouse by adopting the following primers in the step 2):
ApoE-1：GCCTAGCCGAGGGAGAGCCG，
ApoE-2：TGTGACTTGGGAGCTCTGCAGC，
ApoE-3：GCCGCCCCGACTGCATCT。
4. the method of establishing according to claim 2, wherein: the Gfi1 gene point mutation was detected in step 2) using Sanger sequencing.
5. The method of claim 3, wherein: and 2) detecting the ApoE gene by using gel electrophoresis.
6. The method of establishing according to claim 1, wherein: after genotyping F2 generation mice in step 2), immunophenotype of F2 generation mice was analyzed by flow cytometry.
7. The method of establishing according to claim 6, wherein: mouse neutrophils were labeled in said flow cytometry using CD4, CD5, CD8, CD11B, CD11C, CD19, CD25, CD44, CD45, LY6G, and/or NK1.1 antibodies.
8. The method of establishing according to claim 1, wherein: in the breeding process of the model mouse, a male mouse with Gfi1 point mutation and ApoE gene knockout both homozygote, a female mouse with Gfi1 point mutation as heterozygote and ApoE gene knockout as homozygote are selected to breed offspring.
9. The use of the neutrophilic granulocyte deficient atherosclerosis model mouse obtained by the establishment method according to claim 1 for studying interaction between neutrophilic granulocyte deficiency and atherosclerosis.
10. Use according to claim 9, characterized in that: the application of the neutrophile granulocyte deficient atherosclerosis model mouse in researching the effect of neutrophile granulocytes on the formation and repair of atherosclerosis.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810078748.1A CN108271740B (en) | 2018-01-23 | 2018-01-23 | Method for establishing neutrophilic granulocyte-deficient atherosclerosis model mouse |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810078748.1A CN108271740B (en) | 2018-01-23 | 2018-01-23 | Method for establishing neutrophilic granulocyte-deficient atherosclerosis model mouse |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN108271740A CN108271740A (en) | 2018-07-13 |
| CN108271740B true CN108271740B (en) | 2020-01-17 |
Family
ID=62805297
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201810078748.1A Active CN108271740B (en) | 2018-01-23 | 2018-01-23 | Method for establishing neutrophilic granulocyte-deficient atherosclerosis model mouse |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN108271740B (en) |
Families Citing this family (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110742020A (en) * | 2019-09-30 | 2020-02-04 | 中山大学 | Efficient mouse model for hyperlipidemia and atherosclerosis and construction method thereof |
| CN110777167B (en) * | 2019-11-06 | 2021-07-06 | 广州市妇女儿童医疗中心 | Method for constructing mouse model with movement dysfunction phenotype GTPCH enzyme deficiency disease |
| CN113907042B (en) * | 2021-09-30 | 2023-03-24 | 新乡医学院 | Method for constructing neutrophil-loss mouse model |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6156727A (en) * | 1996-09-05 | 2000-12-05 | Uab Research Foundation | Anti-atherosclerotic peptides and a transgenic mouse model of antherosclerosis |
| WO2004074428A2 (en) * | 2003-02-21 | 2004-09-02 | Hvidovre Hospital | Treatment of metabolic disorders |
| CN102517333A (en) * | 2011-12-23 | 2012-06-27 | 上海交通大学医学院附属瑞金医院 | Spontaneous HHcy-related atherosclerosis transgenic mouse animal model and preparation method thereof |
| CN105028994A (en) * | 2015-06-16 | 2015-11-11 | 北京华阜康生物科技股份有限公司 | ApoE mouse formula feed |
-
2018
- 2018-01-23 CN CN201810078748.1A patent/CN108271740B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6156727A (en) * | 1996-09-05 | 2000-12-05 | Uab Research Foundation | Anti-atherosclerotic peptides and a transgenic mouse model of antherosclerosis |
| WO2004074428A2 (en) * | 2003-02-21 | 2004-09-02 | Hvidovre Hospital | Treatment of metabolic disorders |
| CN102517333A (en) * | 2011-12-23 | 2012-06-27 | 上海交通大学医学院附属瑞金医院 | Spontaneous HHcy-related atherosclerosis transgenic mouse animal model and preparation method thereof |
| CN105028994A (en) * | 2015-06-16 | 2015-11-11 | 北京华阜康生物科技股份有限公司 | ApoE mouse formula feed |
Non-Patent Citations (2)
| Title |
|---|
| 大气细颗粒物致动脉粥样硬化小鼠肺脏炎性损伤;柳敏等;《中国公共卫生》;20170131;第33卷(第1期);第98-102页 * |
| 高脂饮食结合球囊损伤快速建立ApoE-/-大鼠动脉粥样硬化;杨炯等;《解放军医学杂志》;20161201;第41卷(第12期);第982-986页 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN108271740A (en) | 2018-07-13 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Chen et al. | Impaired NK1+ T cell development and early IL-4 production in CD1-deficient mice | |
| Barker et al. | Identification of mutations in the COL4A5 collagen gene in Alport syndrome | |
| Morgan et al. | Myeloma aetiology and epidemiology | |
| CN108271740B (en) | Method for establishing neutrophilic granulocyte-deficient atherosclerosis model mouse | |
| Jost et al. | A region on equine chromosome 13 is linked to recurrent airway obstruction in horses | |
| CN108753837B (en) | Construction method of rabbit model with hyperlipidemia or atherosclerosis and sgRNA | |
| CN107580631A (en) | Method for predicting palm oil yield of test oil palm plant and SNP detection kit | |
| Ballesteros-Arias et al. | T cell acute lymphoblastic leukemia as a consequence of thymus autonomy | |
| Le Toriellec et al. | Haploinsufficiency of CDKN1B contributes to leukemogenesis in T-cell prolymphocytic leukemia | |
| Guo et al. | Sequencing reveals population structure and selection signatures for reproductive traits in Yunnan semi-fine wool sheep (Ovis aries) | |
| Cho et al. | Altered microbiota by a high-fat diet accelerates lethal myeloid hematopoiesis associated with systemic SOCS3 deficiency | |
| Yu et al. | A new murine Rpl5 (uL18) mutation provides a unique model of variably penetrant Diamond-Blackfan anemia | |
| Zanella et al. | Fine mapping of loci on BTA2 and BTA26 associated with bovine viral diarrhea persistent infection and linked with bovine respiratory disease in cattle | |
| Marangon et al. | Impact of SNPs/Haplotypes of IL10 and IFNG on the Development of Diffuse Large B‐Cell Lymphoma | |
| Verschoor et al. | SNPs in the bovine IL-10 receptor are associated with somatic cell score in Canadian dairy bulls | |
| Gürses et al. | Identification of the ERV Insertion of APOB gene and deletion of TFB1M gene associated with Lethal Haplotypes of Holstein Cattle Reared in Balikesir province, Türkiye | |
| Fujioka et al. | Targeting retrovirus-derived Rtl8a and 8b causes late onset obesity and neurodevelopmental defects | |
| Bilodeau et al. | Analysis of blood stem cell activity and cystatin gene expression in a mouse model presenting a chromosomal deletion encompassing Csta and Stfa2l1 | |
| Drögemüller et al. | Genetic analysis of syndactyly in German Holstein cattle | |
| CN112961864A (en) | Mutant ASXL3 gene and application | |
| Ek et al. | Generation of a multi-locus chicken introgression line to study the effects of genetic interactions on metabolic phenotypes in chickens | |
| Korstanje et al. | Mapping of microsatellite loci and association of aorta atherosclerosis with LG VI markers in the rabbit | |
| CN112522312B (en) | WKH rat model construction method | |
| Prakash et al. | Distribution analysis of single nucleotide polymorphisms related to feet and legs and their association with lameness in Vrindavani cattle | |
| CN111808859B (en) | gRNA of WAS gene 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 | ||
| GR01 | Patent grant |