CN110771573A - PirB基因敲入的小鼠动物模型及其构建方法 - Google Patents
PirB基因敲入的小鼠动物模型及其构建方法 Download PDFInfo
- Publication number
- CN110771573A CN110771573A CN201911120817.1A CN201911120817A CN110771573A CN 110771573 A CN110771573 A CN 110771573A CN 201911120817 A CN201911120817 A CN 201911120817A CN 110771573 A CN110771573 A CN 110771573A
- Authority
- CN
- China
- Prior art keywords
- pirb
- mouse
- gene
- animal model
- grna1
- 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
- 241000699666 Mus <mouse, genus> Species 0.000 title claims abstract description 60
- 238000010171 animal model Methods 0.000 title claims abstract description 29
- 101100128415 Mus musculus Lilrb3 gene Proteins 0.000 title claims abstract description 17
- 101100236064 Rattus norvegicus Lilrb3l gene Proteins 0.000 title claims abstract 16
- 238000010276 construction Methods 0.000 title abstract description 10
- 241000699670 Mus sp. Species 0.000 claims abstract description 36
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 32
- 238000011746 C57BL/6J (JAX™ mouse strain) Methods 0.000 claims abstract description 6
- 108020004414 DNA Proteins 0.000 claims description 18
- 108091033409 CRISPR Proteins 0.000 claims description 14
- 238000003198 gene knock in Methods 0.000 claims description 13
- 238000000034 method Methods 0.000 claims description 13
- 238000012163 sequencing technique Methods 0.000 claims description 13
- 230000008685 targeting Effects 0.000 claims description 10
- 238000002105 Southern blotting Methods 0.000 claims description 9
- 239000013598 vector Substances 0.000 claims description 9
- 235000013601 eggs Nutrition 0.000 claims description 5
- 238000010354 CRISPR gene editing Methods 0.000 claims description 4
- 238000002347 injection Methods 0.000 claims description 4
- 239000007924 injection Substances 0.000 claims description 4
- 108010009861 CCTAGG-specific type II deoxyribonucleases Proteins 0.000 claims description 3
- 108010005054 Deoxyribonuclease BamHI Proteins 0.000 claims description 3
- 238000005516 engineering process Methods 0.000 claims description 3
- 238000009399 inbreeding Methods 0.000 claims description 3
- 230000013011 mating Effects 0.000 claims description 3
- 241001465754 Metazoa Species 0.000 claims description 2
- 238000009395 breeding Methods 0.000 claims description 2
- 230000001488 breeding effect Effects 0.000 claims description 2
- 230000007935 neutral effect Effects 0.000 claims description 2
- 230000006870 function Effects 0.000 abstract description 6
- 238000011160 research Methods 0.000 abstract description 4
- 210000000056 organ Anatomy 0.000 abstract description 2
- 201000010099 disease Diseases 0.000 abstract 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 abstract 1
- 238000009396 hybridization Methods 0.000 abstract 1
- 101000984191 Mus musculus Leukocyte immunoglobulin-like receptor subfamily B member 3 Proteins 0.000 description 73
- 239000000047 product Substances 0.000 description 16
- 239000000523 sample Substances 0.000 description 12
- 210000002919 epithelial cell Anatomy 0.000 description 8
- 108700028369 Alleles Proteins 0.000 description 7
- 230000002401 inhibitory effect Effects 0.000 description 7
- 108020003175 receptors Proteins 0.000 description 7
- 102000005962 receptors Human genes 0.000 description 7
- 210000004027 cell Anatomy 0.000 description 6
- 239000012634 fragment Substances 0.000 description 6
- 238000001179 sorption measurement Methods 0.000 description 6
- 238000000137 annealing Methods 0.000 description 5
- 238000010586 diagram Methods 0.000 description 5
- 210000000987 immune system Anatomy 0.000 description 5
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 4
- 238000012408 PCR amplification Methods 0.000 description 4
- 230000003321 amplification Effects 0.000 description 4
- 238000006243 chemical reaction Methods 0.000 description 4
- 239000007788 liquid Substances 0.000 description 4
- 238000003199 nucleic acid amplification method Methods 0.000 description 4
- 102000004169 proteins and genes Human genes 0.000 description 4
- 210000001519 tissue Anatomy 0.000 description 4
- 210000003719 b-lymphocyte Anatomy 0.000 description 3
- 230000001537 neural effect Effects 0.000 description 3
- 230000008569 process Effects 0.000 description 3
- 208000024827 Alzheimer disease Diseases 0.000 description 2
- 238000011740 C57BL/6 mouse Methods 0.000 description 2
- 102000053602 DNA Human genes 0.000 description 2
- 108010067770 Endopeptidase K Proteins 0.000 description 2
- 108020005004 Guide RNA Proteins 0.000 description 2
- 108060003951 Immunoglobulin Proteins 0.000 description 2
- 241000700605 Viruses Species 0.000 description 2
- 230000004913 activation Effects 0.000 description 2
- 230000033228 biological regulation Effects 0.000 description 2
- 210000003169 central nervous system Anatomy 0.000 description 2
- 210000000349 chromosome Anatomy 0.000 description 2
- 230000001086 cytosolic effect Effects 0.000 description 2
- 230000029087 digestion Effects 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 238000001962 electrophoresis Methods 0.000 description 2
- 239000003480 eluent Substances 0.000 description 2
- 238000000605 extraction Methods 0.000 description 2
- 102000018358 immunoglobulin Human genes 0.000 description 2
- 239000003112 inhibitor Substances 0.000 description 2
- 238000011813 knockout mouse model Methods 0.000 description 2
- 230000007246 mechanism Effects 0.000 description 2
- 239000012528 membrane Substances 0.000 description 2
- 210000000653 nervous system Anatomy 0.000 description 2
- 210000002241 neurite Anatomy 0.000 description 2
- 239000013600 plasmid vector Substances 0.000 description 2
- 238000010008 shearing Methods 0.000 description 2
- 239000008223 sterile water Substances 0.000 description 2
- 239000006228 supernatant Substances 0.000 description 2
- 230000003956 synaptic plasticity Effects 0.000 description 2
- 238000001890 transfection Methods 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 1
- GNFTZDOKVXKIBK-UHFFFAOYSA-N 3-(2-methoxyethoxy)benzohydrazide Chemical compound COCCOC1=CC=CC(C(=O)NN)=C1 GNFTZDOKVXKIBK-UHFFFAOYSA-N 0.000 description 1
- QRXMUCSWCMTJGU-UHFFFAOYSA-N 5-bromo-4-chloro-3-indolyl phosphate Chemical compound C1=C(Br)C(Cl)=C2C(OP(O)(=O)O)=CNC2=C1 QRXMUCSWCMTJGU-UHFFFAOYSA-N 0.000 description 1
- 208000014912 Central Nervous System Infections Diseases 0.000 description 1
- 108020004705 Codon Proteins 0.000 description 1
- 238000007400 DNA extraction Methods 0.000 description 1
- 238000013382 DNA quantification Methods 0.000 description 1
- 101001065501 Escherichia phage MS2 Lysis protein Proteins 0.000 description 1
- 108700024394 Exon Proteins 0.000 description 1
- 108090000288 Glycoproteins Proteins 0.000 description 1
- 102000003886 Glycoproteins Human genes 0.000 description 1
- 101000876511 Homo sapiens General transcription and DNA repair factor IIH helicase subunit XPD Proteins 0.000 description 1
- 101000984189 Homo sapiens Leukocyte immunoglobulin-like receptor subfamily B member 2 Proteins 0.000 description 1
- 101000616778 Homo sapiens Myelin-associated glycoprotein Proteins 0.000 description 1
- 108050006430 Immunoglobulin-like domains Proteins 0.000 description 1
- 102000016844 Immunoglobulin-like domains Human genes 0.000 description 1
- OUYCCCASQSFEME-QMMMGPOBSA-N L-tyrosine Chemical compound OC(=O)[C@@H](N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-QMMMGPOBSA-N 0.000 description 1
- 241000713666 Lentivirus Species 0.000 description 1
- 102100025583 Leukocyte immunoglobulin-like receptor subfamily B member 2 Human genes 0.000 description 1
- 108700018351 Major Histocompatibility Complex Proteins 0.000 description 1
- 241001529936 Murinae Species 0.000 description 1
- 101100027996 Mus musculus Omg gene Proteins 0.000 description 1
- 102000006386 Myelin Proteins Human genes 0.000 description 1
- 108010083674 Myelin Proteins Proteins 0.000 description 1
- 102000010410 Nogo Proteins Human genes 0.000 description 1
- 108010077641 Nogo Proteins Proteins 0.000 description 1
- 239000004677 Nylon Substances 0.000 description 1
- 102000001253 Protein Kinase Human genes 0.000 description 1
- 108091081024 Start codon Proteins 0.000 description 1
- 208000006011 Stroke Diseases 0.000 description 1
- 210000001744 T-lymphocyte Anatomy 0.000 description 1
- 108700019146 Transgenes Proteins 0.000 description 1
- 102100023935 Transmembrane glycoprotein NMB Human genes 0.000 description 1
- 208000030886 Traumatic Brain injury Diseases 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 238000000246 agarose gel electrophoresis Methods 0.000 description 1
- 150000001413 amino acids Chemical class 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 210000001130 astrocyte Anatomy 0.000 description 1
- 210000003050 axon Anatomy 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000008499 blood brain barrier function Effects 0.000 description 1
- 210000001218 blood-brain barrier Anatomy 0.000 description 1
- 210000004556 brain Anatomy 0.000 description 1
- 239000000872 buffer Substances 0.000 description 1
- 239000007853 buffer solution Substances 0.000 description 1
- 230000020411 cell activation Effects 0.000 description 1
- 230000024245 cell differentiation Effects 0.000 description 1
- 230000006727 cell loss Effects 0.000 description 1
- 230000001413 cellular effect Effects 0.000 description 1
- 238000003776 cleavage reaction Methods 0.000 description 1
- 210000001151 cytotoxic T lymphocyte Anatomy 0.000 description 1
- 210000004443 dendritic cell Anatomy 0.000 description 1
- 230000001419 dependent effect Effects 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 230000018109 developmental process Effects 0.000 description 1
- 238000010828 elution Methods 0.000 description 1
- 238000001976 enzyme digestion Methods 0.000 description 1
- 230000008472 epithelial growth Effects 0.000 description 1
- 235000019441 ethanol Nutrition 0.000 description 1
- 238000003209 gene knockout Methods 0.000 description 1
- 238000010363 gene targeting Methods 0.000 description 1
- 238000003205 genotyping method Methods 0.000 description 1
- 210000004602 germ cell Anatomy 0.000 description 1
- 210000003714 granulocyte Anatomy 0.000 description 1
- 210000000020 growth cone Anatomy 0.000 description 1
- 238000010438 heat treatment Methods 0.000 description 1
- 210000003958 hematopoietic stem cell Anatomy 0.000 description 1
- 210000003630 histaminocyte Anatomy 0.000 description 1
- 230000002209 hydrophobic effect Effects 0.000 description 1
- 230000003832 immune regulation Effects 0.000 description 1
- 230000028993 immune response Effects 0.000 description 1
- 239000012535 impurity Substances 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 230000005764 inhibitory process Effects 0.000 description 1
- 230000003834 intracellular effect Effects 0.000 description 1
- 210000002540 macrophage Anatomy 0.000 description 1
- 108091005450 macrophage integrins Proteins 0.000 description 1
- 108020004999 messenger RNA Proteins 0.000 description 1
- 238000000520 microinjection Methods 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 238000010172 mouse model Methods 0.000 description 1
- 210000005012 myelin Anatomy 0.000 description 1
- 210000000066 myeloid cell Anatomy 0.000 description 1
- 210000000822 natural killer cell Anatomy 0.000 description 1
- 210000002569 neuron Anatomy 0.000 description 1
- 230000014511 neuron projection development Effects 0.000 description 1
- 210000000440 neutrophil Anatomy 0.000 description 1
- 229920001778 nylon Polymers 0.000 description 1
- 230000009437 off-target effect Effects 0.000 description 1
- 238000007500 overflow downdraw method Methods 0.000 description 1
- 230000001575 pathological effect Effects 0.000 description 1
- 230000037361 pathway Effects 0.000 description 1
- 239000013612 plasmid Substances 0.000 description 1
- 230000008929 regeneration Effects 0.000 description 1
- 238000011069 regeneration method Methods 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 230000008844 regulatory mechanism Effects 0.000 description 1
- 108091008146 restriction endonucleases Proteins 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- 230000007017 scission Effects 0.000 description 1
- 239000000243 solution Substances 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 230000020382 suppression by virus of host antigen processing and presentation of peptide antigen via MHC class I Effects 0.000 description 1
- 230000000946 synaptic effect Effects 0.000 description 1
- 108091007466 transmembrane glycoproteins Proteins 0.000 description 1
- OUYCCCASQSFEME-UHFFFAOYSA-N tyrosine Natural products OC(=O)C(N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-UHFFFAOYSA-N 0.000 description 1
- 238000001262 western blot Methods 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
-
- 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
- C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
- C07K14/70503—Immunoglobulin superfamily
-
- 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
-
- 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
- C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
- C12N15/90—Stable introduction of foreign DNA into chromosome
- C12N15/902—Stable introduction of foreign DNA into chromosome using homologous recombination
- C12N15/907—Stable introduction of foreign DNA into chromosome using homologous recombination in mammalian 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
- A01K2217/072—Animals genetically altered by homologous recombination maintaining or altering function, i.e. knock in
-
- 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
-
- 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
- A01K2267/0387—Animal model for diseases of the immune system
-
- 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
-
- 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
- C12N2800/30—Vector systems comprising sequences for excision in presence of a recombinase, e.g. loxP or FRT
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Genetics & Genomics (AREA)
- Engineering & Computer Science (AREA)
- Chemical & Material Sciences (AREA)
- Zoology (AREA)
- Organic Chemistry (AREA)
- Biotechnology (AREA)
- General Health & Medical Sciences (AREA)
- Wood Science & Technology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- Biomedical Technology (AREA)
- Molecular Biology (AREA)
- Biochemistry (AREA)
- Biophysics (AREA)
- Physics & Mathematics (AREA)
- Immunology (AREA)
- Cell Biology (AREA)
- Plant Pathology (AREA)
- Environmental Sciences (AREA)
- Microbiology (AREA)
- Veterinary Medicine (AREA)
- Animal Husbandry (AREA)
- Animal Behavior & Ethology (AREA)
- Biodiversity & Conservation Biology (AREA)
- Mycology (AREA)
- Toxicology (AREA)
- Gastroenterology & Hepatology (AREA)
- Medicinal Chemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
本发明公开了PirB基因敲入的小鼠动物模型,该模型包括确定PirB基因待敲入的特异性靶位点的gRNA1和gRNA2,将PirB基因敲入C57BL/6J小鼠的ROSA26基因的内含子1内,gRNA1的基因序列如SEQ ID NO.1所示,gRNA2的基因序列如SEQ ID NO.2所示。本发明还具体公开了上述小鼠动物模型的构建方法,本发明的小鼠动物模型对于PirB基因功能的研究和在体验证提供了良好的基础。通过PirB敲入动物模型与不同类型Cre小鼠的杂交,可以用于研究PirB在不同器官或不同细胞类型或不同疾病模型的功能。
Description
技术领域:
本发明属于遗传学和生物技术领域,具体涉及PirB基因敲入的小鼠动物模型,还涉及上述小鼠动物模型的构建方法。
背景技术
鼠源成对免疫球蛋白样受体B(paired immunoglobulin-like receptor B,PirB),是人类免疫球蛋白样受体B2(human leukocyte immunoglobulin(Ig)-likereceptor B2,LILRB2)的同源基因,PirB基因(NCBI reference sequence:NM_011095.2)位于小鼠的7号染色体近端,总大小为8.97kb,编码841个氨基酸,目前鉴定出15个外显子,外显子1起始密码为ATG,外显子15的终止密码子是TGA(转录本:Pirb-201ENSMUST00000078451.6)。PirB蛋白属于I型跨膜糖蛋白,包含由六个免疫球蛋白样结构域(domain,D)组成(D1-D6)的胞外段,一个疏水的跨膜段,三个免疫受体酪氨酸依赖的抑制序列(immunoreceptortyrosinebased inhibitory motifs,ITIMs)和一个ITIMs样序列组成的胞内段。理论上讲,PirB的分子量约为92kD,但是Western-blot分析中,PirB经常位于105kD处,因为PirB是糖蛋白。以往研究发现,PirB在免疫***和中枢调节中均发挥重要作用。
在免疫***中,PirB在许多造血细胞都有表达,包括B细胞、肥大细胞、巨噬细胞、粒细胞和树突细胞,但是在胸腺细胞、成熟的T细胞和自然杀伤细胞不表达,另外PirB在髓细胞和B细胞的表达随细胞分化和激活增加。在免疫***,PirB作为主要组织相容性复合物1(class I major histocompatibility complex,MHC1)的受体,广泛参与免疫调节,包括中性粒细胞和巨噬细胞整合素通路、细胞毒性T淋巴细胞激活、B淋巴细胞激活和体液—细胞免疫应答等。PirB的前两个细胞外免疫球蛋白结构域(D1-D2)介导MHCI和PirB的结合。
在中枢神经***(central nervous system,CNS),PirB在许多脑区包括皮层、海马、小脑和嗅球都有表达,但在成年脊髓不表达。在细胞层面,PirB不仅表达于神经元,也表达于星形胶质细胞。在许多病理条件下,如脑外伤、中风和CNS感染,PirB的mRNA和蛋白表达水平显著增加。在CNS,PirB是髓鞘抑制因子Nogo-A、MAG、OMgp的受体,参与神经元突起芽发和生长锥塌陷,抑制神经元轴突再生和突触可塑性。PirB的细胞外免疫球蛋白结构域(D3-D6)介导了PirB与NogoA的结合。近年来关于阿尔茨海默病(Alzheimer’s disease,AD)研究还发现,PirB是Aβ42寡聚体的高亲和力受体,亲和力达到纳摩尔水平。PirB的前两个细胞外免疫球蛋白结构域(D1-D2)介导了Aβ42寡聚体和PirB的相互作用,导致丝切蛋白(cofilin)信号通路增强。免疫组织化学染色结果发现,PirB在轴突生长锥表达,位于富含肌动蛋白的前缘和synapsin免疫阳性的囊泡中,在神经元突起的表达呈点状分布。文献表明,PirB表达随年龄增加,特别是在老龄认知损伤的小鼠海马中,PirB能够抑制轴突再生和突触可塑性。研究表明小鼠Aβ寡聚体对海马长时程增强的破坏作用需要PirB的参与,在AD转基因模型中,PirB不仅参与成年小鼠记忆缺失,而且介导幼年小鼠视皮层突触可塑性的丢失。这些研究提示我们,PirB参与突触可塑性,抑制PirB可能对AD起到治疗效应。但是在CNS,PirB的功能和下游抑制性信号通路仍然未被阐明,因此迫切需要PirB的细胞和动物模型。
目前对于PirB基因功能的研究,多采用可溶性的PirB的胞外段(PirBextracellular peptide,PEP)和抑制剂,或者采用慢病毒转染。前者受到是否能够透过血脑屏障和抑制剂效率的影响,后者慢病毒转染在原代细胞和在体的转染效率比较低,使研究PirB的功能受到极大的限制。为解决这些问题,我们通过CRISPR/Cas9技术建立了PirB基因敲入小鼠,将PirB基因敲入C57BL/6J的ROSA26位点,为研究PirB在免疫***或者神经***的作用和机制提供了很好的工具。
发明内容
本发明的目的在于提供PirB基因敲入的小鼠动物模型,该模型能够帮助我们研究PirB基因在免疫***和神经***的功能以及下游的调节机制。
本发明的另一目的在于提供上述小鼠动物模型的构建方法。
本发明所采用的第一种技术方案是:PirB基因敲入的小鼠动物模型,包括确定PirB基因的待敲入的特异性靶位点gRNA1和gRNA2,将PirB基因敲入C57BL/6J小鼠的ROSA26基因的内含子1内,所述gRNA1的基因序列如SEQ ID NO.1所示,gRNA2的基因序列如SEQ IDNO.2所示。
本发明所采用的第二种技术方案为:PirB基因敲入的小鼠动物模型的构建方法,具体按照以下步骤实施:
步骤1、基于CRISPR/Cas9技术构建针对C57BL/6J小鼠ROSA26基因的特异性gRNA1和gRNA2,gRNA1的基因序列如SEQ ID NO.1所示,gRNA2的基因序列如SEQ ID NO.2所示;
步骤2、构建“CAG promoter-loxP-Stop-loxP-Kozak-mouse PirbCDS-polyA”基因盒打靶载体,将打靶载体进行线性化处理;
步骤3、步骤2中将含有loxP位点打靶载体、步骤1中有活性的gRNA1和gRNA2与Cas9蛋白共同注射进入***小鼠受精卵中,获得F0代小鼠;
步骤4、将步骤3中性成熟的阳性F0小鼠分别与野生型鼠交配繁一代,获得F1代杂合子小鼠,通过PCR、测序和Southern杂交确定动物基因型;
步骤5、将步骤4获得的F1代杂合子小鼠近交获得F2代纯合子小鼠,即为本发明构建的一种PirB基因敲入的小鼠动物模型。
本发明所采用第二种技术方案的特点还在于,
步骤1中得到gRNA1和gRNA2后分别与trancrRNA在25℃孵育10min形成二级结构。
步骤3中gRNA1、gRNA2的浓度均为2~10pmol/ul,Cas9蛋白的注射浓度为30~100ng/μL。
步骤4中Southern杂交采用BamHI和AvrII核酸内切酶切割F1代杂合子小鼠的DNA。
本发明的有益效果是:本发明提供了PirB基因敲入的小鼠动物模型及其构建方法,本发明的小鼠动物模型对于PirB基因功能的研究和在体验证提供了良好的基础。分离自PirB基因敲入小鼠的细胞还可以用于研究PirB发挥调节作用的下游机制。通过PirB敲入动物模型与不同类型Cre小鼠的杂交,可以用于研究AD不同器官或不同细胞类型PirB的调节作用。
附图说明
图1为本发明PirB基因敲入的小鼠***PirB基因CDS和loxP位点的打靶质粒载体的构建图;
图2为本发明PirB基因敲入的小鼠模型的构建方法的流程图;
图3为本发明F1代PirB敲入的小鼠的基因型PCR结果鉴定电泳图;
图4为本发明F1代PirB敲入的小鼠验证PirB基因敲入效果的测序峰图;
图5为本发明F1代PirB基因敲入的小鼠进行Southern鉴定的方法示意图;
图6为本发明F1代PirB基因敲入的小鼠进行Southern鉴定的结果图。
具体实施方式
下面结合附图和具体实施方式对发明作进一步阐述。
本发明构建了PirB基因敲入的小鼠动物模型,将PirB基因敲入C57BL/6J小鼠的ROSA26基因的内含子1内。具体来说,构建一个CAG promoter-loxP-Stop-loxP-Kozak-mouse PirB CDS-polyA的基因盒,将其克隆进入ROSA26基因的内含子1中。
ROSA26基因(NCBI Reference Sequence:NR_011095.2)位于小鼠的七号染色体。
本发明PirB基因敲入的小鼠动物模型的构建方法,具体按照以下步骤具体实施:
步骤1、根据小鼠ROSA26基因(Gene Bank:NR_027008.1))序列,利用Cas-Desigher软件在1号内含子设计gRNA靶序列,并搜索小鼠DBM-DB基因组数据库基因,采用CRISPR脱靶效应软件Cas-OFFinder检测潜在的脱靶位点:最终选取两个gRNA,gRNA1的基因序列如SEQID NO.1所示,gRNA2的基因序列如SEQ ID NO.2所示:
SEQ ID NO.1:GGCAGGCTTAAAGGCTAACCTGG
SEQ ID NO.2:CTCCAGTCTTTCTAGAAGATGGG
将gRNA1和gRNA2分别与trancrRNA在25℃孵育10min形成二级结构;
步骤2、利用C57BL/6小鼠文库BAC克隆,通过PCR扩增获得PirB基因CDS的同源臂,采用in-fusion方法构建含有CAG promoter-loxP-Stop-loxP-Kozak-mouse PirB CDS-polyA的基因盒的质粒打靶载体,通过酶切、PCR及测序对打靶质粒载体进行验证,图1为构建好的PirB基因打靶载体的示意图。
步骤3、PMSG处理C57BL/6雌性小鼠(6周龄,平均体重20g),46h后注射hCG,与雄性小鼠合笼交配,次日取受精卵进行显微注射,将步骤1的gRNA1、gRNA2、Cas9蛋白以及线性化后的打靶载体一起注射到受精卵中,取注射后存活的受精卵移植到假孕母鼠体内,产出小鼠,即F0代小鼠。
上述步骤中gRNA1、gRNA2的浓度均为2~10pmol/ul,Cas9蛋白的注射浓度为30~100ng/μL,Cas9蛋白购自New England Biolabs,货号为M0386M。
步骤4、提取F0代小鼠尾部DNA,PCR扩增产物测序,鉴定是否为嵌合体。
本发明用于F0代小鼠PirB基因PCR鉴定的特异性引物如下:
PCR Primers 1(Annealing Temperature 60.0℃):
5’arm forward primer(F1):
5’-TACGCCACAGGGAGTCCAAGAATG-3’
5’KI reverse primer(R1):
5’-GATGGGGAGAGTGAAGCAGAACG-3’
PCR Primers 2(Annealing Temperature 60.0℃):
3’KI forward primer(F2):
5’-CTGCTGTCCATTCCTTATTCCATAG-3’
3’arm reverse primer(R2):
5’-CTGGAAATCAGGCTGCAAATCTC-3’。
Primers1对应的扩增产物大小为2.7kb,Primers2对应的扩增产物为2.5kb。
用于F0代小鼠测序的引物如下:
Sequencing Primer for PCR product 1:
5’Sequence primer(F3):
5’-CACTTGCTCTCCCAAAGTCGCTC-3’
Sequencing Primer for PCR product 2:
3’Sequence primer(R3):5’-ATACTCCGAGGCGGATCACAA-3’
步骤4、鉴定为PirB基因敲入的F0代雄性小鼠7周龄、雌性小鼠6周龄分别与野生型异性小鼠交配获得F1代杂合子小鼠,图2为本发明F1代杂合子小鼠PirB基因敲除小鼠的流程图,小鼠出生后20天进行PCR鉴定,若有阳性小鼠出生,则表示转基因已经整合到生殖细胞。
(1)通过PCR方法对获得的F1代小鼠进行基因型的鉴定:
(A)DNA的提取:
方法1:采用TaKaRaMiniBEST Universal Genomic DNA Extraction kit(Ver.5.0_Code No.9765)来获得高纯度的基因组DNA。
步骤a.每只小鼠剪下一段尾巴(2~5mm),放入离心管中,加入180μLBufferGL,20μL蛋白激酶K和10μLRNase A。
步骤b.将步骤a离心管内于56℃孵育过夜。
步骤c.过夜后12000rpm离心2min去除杂质。
步骤d.加入200μLBuffer GB和200μL无水乙醇,充分混匀。
步骤e.将吸附柱放在收集管上,将步骤d得到的样品加到吸附柱里,12000rpm离心2min,弃掉收集管中液体。
步骤f.弃去液体后在吸附柱中加入500μLBuffer WA,12000rpm离心1min,弃掉收集管中液体。
步骤g.在收集管中加入700μL Buffer WB,12000rpm离心1min。弃掉收集管中液体。
注意:BufferWB需要与100%乙醇预混,沿管壁加入Buffer WB冲走残余的盐。
步骤h.重复步骤g一次。
步骤i.将步骤h的吸附柱放入收集管,12000rpm离心2min。
步骤j.吸附柱放到一个新的1.5mL的离心管,在吸附柱膜中央加入50~200μL灭菌水或者洗脱液,停留5min。(将无菌水或者洗脱液加热到65℃能够增加洗脱的得率)。
步骤i.基因组DNA定量,洗脱的基因组DNA可以通过电泳鉴定。
方法2:一种低成本基因组DNA的粗提方法:
步骤a.每只小鼠剪下一段尾巴(2-5mm),放入离心管中,加入100μL尾部消化缓冲液,注意不要剪尾太长;
步骤b.将离心管及其内容物于56℃孵育过夜;
步骤c.过夜后将离心管于98℃孵育13min,使蛋白酶K失活;
步骤d.在微型离心机以最大转速旋转15min,上清直接用于PCR体系(每50μLPCR体系加入上清2μL)。
尾消化缓冲液成分:
50mM KCl
10mM Tris-HCl(pH9.0)
0.1%TritonX-100
0.4mg/mL Proteinase K
(B)长链PCR反应:
长链引物:
PCR Primers 1(Annealing Temperature 60.0℃):扩增片段:2.7kb
5’arm forward primer(F1):
5’-TACGCCACAGGGAGTCCAAGAATG-3’
5’KI reverse primer(R1):
5’-GATGGGGAGAGTGAAGCAGAACG-3’
PCR Primers 2(Annealing Temperature 60.0℃):扩增片段:2.5kb
3’KI forward primer(F2):
5’-CTGCTGTCCATTCCTTATTCCATAG-3’
3’arm reverse primer(R2):
5’-CTGGAAATCAGGCTGCAAATCTC-3’
PirB基因敲除型有上述两条扩增片段,野生型等位基因没有扩增产物。
PCR Mix:
反应条件:
PCR扩增产物的电泳鉴定结果如图3所示,从图3中可以看出,6、8、9号为PirB基因敲入小鼠在2.7kb、2.5kb有特异性扩增产物,WT小鼠PCR产物没有这两个扩增产物。
(C)短链PCR反应,引物如下:
Primers(Annealing Temperature 60.0℃):
Forward primer(F4):5’-AACAATCAGGCTGCCGAATCTG-3’
Reverse primer(R4):5’-CTTTATTAGCCAGAAGTCAGATGC-3’
Expected PCR Product:
Targeted allele:344bp
PirB基因敲入型小鼠能够扩增出344bp的产物,而野生型没有。
PCR体系:
反应条件:
(2)F1代小鼠测序:
通过PCR扩增后测序鉴定小鼠基因型,如图4所示,为6号PirB基因敲入小鼠测序峰图,PCR所用引物如下:
Sequencing Primer for PCR product 1:
5’Sequence primer(F3):
5’-CACTTGCTCTCCCAAAGTCGCTC-3’
Sequencing Primer for PCR product 2:
3’Sequence primer(R3):
5’-ATACTCCGAGGCGGATCACAA-3’
(3)Southern杂交检测F1代小鼠基因型:
采用BamHI和AvrII核酸内切酶切割DNA分子,切割示意图如图5。具体的Southern杂交检测过程如下:
步骤a、提取F1代小鼠尾部DNA;
步骤b、制备探针,通过PCR方法将Dig-dUTP渗入到步骤a的DNA分子中;
F1代小鼠southern印记的5’探针引物如下:
Primers for 5’Probe:
5’Probe forward primer:
5’-AAACGTGGAGTAGGCAATACCCAGG-3’
5’Probe reverse primer:
5’-AAAGAAGGGTCACCTCAGTCTCCCT-3’
Primers for 3’Probe:
3’Probe forward primer:
5’-TTCTGGGCAGGCTTAAAGGCTAAC-3’
3’Probe reverse primer:
5’-AGGAGCGGGAGAAATGGATATGAAG-3’
Southern印记的目标片段大小如下:
5’Probe-BamHI:5.83kb-WT,4.80kb-MT
3’Probe-AvrII:5.27kb-WT,11.12kb-MT。
步骤c、采用限制性内切酶BamHI和AvrII对DNA进行切割;琼脂糖凝胶电泳分离DNA;
步骤d、将DNA转到尼龙膜上;
步骤e、探针变性后,与DNA分子进行杂交,NBT/BCIP化学显色法显色,拍照观察。Southern杂交结果如图6所示,可以看到:6、8、9号PirB基因敲除小鼠如果被BamHI切割,在5.83kb和3.56kb有两个片段,WT小鼠只在5.83kb处有一个切割片段,如果被AvrII切割,PirB基因敲除小鼠在11.12kb和5.27kb有两个片段,WT小鼠只在5.27kb处有一个切割片段。
步骤6、将F1代杂合子小鼠近交获得F2代纯合子PirB基因敲入小鼠,即为本发明所述小鼠PirB基因敲入的动物模型。
将本发明获得的动物模型F2代纯合子小鼠与同品系小鼠组织/细胞特异性Cre表达的小鼠杂交,获得F3代小鼠,可以实现在不同组织或者细胞类型中敲入PirB基因。
对F3代小鼠进行基因型的鉴定:含有无(PirB-cWT)、一个(PirB-cHET)或者两个(PirB-cKI)PirB基因敲入的小鼠,PirB基因敲入到特异性组织或细胞中。其中PirB-cWT小鼠表达Cre,但不含有loxP-PirB等位基因,PirB-cHET and PirB-cKI小鼠表达Cre并分别含有一个或者两个loxP-PirB等位基因。
其中F3代杂交小鼠纯合子/杂合子鉴定所用的PCR引物如下:
F3:5’-CACTTGCTCTCCCAAAGTCGCTC-3’
R3:5’-ATACTCCGAGGCGGATCACAA-3’
F5:5’-GCATCTGACTTCTGGCTAATAAAG-3’
Homozygotes:644bp
Heterozygotes:644bp/453bp
Wildtype allele:453bp
组织特异性的基因敲入也可以通过加入另一对引物,用PCR来验证:
PCR for constitutive KI allele:
F6:5’-GGCAACGTGCTGGTTATTGTG-3’
R6:5’-GGCTGTACTCTGAGGATAGGCTTAG-3’
F7:5’-AGATCTGCAAGCTAATTCCTGC-3’
With CKI:1180bp/215bp
Constitutive KI allele:303bp
注意:如果DNA的样本不够纯,或者PCR的延伸时间不够长,可能扩增不出来1180bp的产物。
Cre阳性PCR鉴定所需的引物如下:
Forward:5’-GAACGCACTGATTTCGACCA-3’
Reverse:5’-GCTAACCAGCGTTTTCGTTC-3’
Creamplicon:204bp。
序列表
<110> 西安医学院
<120> PirB基因敲入的小鼠动物模型及其构建方法
<130> 2019
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 23
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 1
ggcaggctta aaggctaacc tgg 23
<210> 2
<211> 23
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 2
ctccagtctt tctagaagat ggg 23
Claims (5)
1.PirB基因敲入的小鼠动物模型,其特征在于,所述小鼠动物模型包括确定PirB基因的待敲入的特异性靶位点gRNA1和gRNA2,将PirB基因敲入C57BL/6J小鼠的ROSA26基因的内含子1内,所述gRNA1的基因序列如SEQ ID NO.1所示,所述gRNA2的基因序列如SEQ ID NO.2所示。
2.一种权利要求1所述的PirB基因敲入的小鼠动物模型的构建方法,其特征在于,按照以下步骤进行实施:
步骤1、基于CRISPR/Cas9技术构建针对C57BL/6J小鼠ROSA26基因内含子1的特异性gRNA1和gRNA2,所述gRNA1的基因序列如SEQ ID NO.1所示,所述gRNA2的基因序列如SEQ IDNO.2所示;
步骤2、构建“CAG promoter-loxP-Stop-loxP-Kozak-mouse PirbCDS-polyA”基因盒打靶载体,将打靶载体进行线性化处理;
步骤3、步骤2中将含有loxP位点打靶载体、步骤1中有活性的gRNA1和gRNA2和Cas9蛋白共同注射进入***小鼠受精卵中,获得F0代小鼠;
步骤4、将步骤3中性成熟的阳性F0小鼠分别与野生型鼠交配繁一代,获得F1代杂合子小鼠,通过PCR、测序和Southern杂交鉴定动物基因型;
步骤5、将步骤4获得的F1代杂合子小鼠近交获得F2代纯合子小鼠,即为本发明构建的一种PirB基因敲入的小鼠动物模型。
3.根据权利要求2所述的PirB基因敲入的小鼠动物模型的构建方法,其特征在于,所述步骤1中得到gRNA1和gRNA2后需要将gRNA1和gRNA2分别与trancrRNA在25℃孵育10min形成二级结构。
4.根据权利要求2所述的PirB基因敲入的小鼠动物模型的构建方法,其特征在于,所述步骤3中gRNA1、gRNA2的浓度均为2~10pmol/ul,Cas9蛋白的注射浓度为30~100ng/μL。
5.根据权利要求2所述的PirB基因敲入的小鼠动物模型的构建方法,其特征在于,所述步骤4中Southern杂交采用BamHI和AvrII核酸内切酶切割F1代杂合子小鼠的DNA。
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN201911120817.1A CN110771573B (zh) | 2019-11-15 | 2019-11-15 | PirB基因敲入的小鼠动物模型及其构建方法 |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN201911120817.1A CN110771573B (zh) | 2019-11-15 | 2019-11-15 | PirB基因敲入的小鼠动物模型及其构建方法 |
Publications (2)
Publication Number | Publication Date |
---|---|
CN110771573A true CN110771573A (zh) | 2020-02-11 |
CN110771573B CN110771573B (zh) | 2021-08-27 |
Family
ID=69391500
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN201911120817.1A Expired - Fee Related CN110771573B (zh) | 2019-11-15 | 2019-11-15 | PirB基因敲入的小鼠动物模型及其构建方法 |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN110771573B (zh) |
Cited By (6)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN111500639A (zh) * | 2020-04-15 | 2020-08-07 | 徐州医科大学 | Stat3线粒体定位条件性基因敲入小鼠模型的构建方法 |
CN111718932A (zh) * | 2020-06-08 | 2020-09-29 | 中国人民解放军军事科学院军事医学研究院 | 一种新型的基因编辑动物生物反应器制备方法及应用 |
CN112266931A (zh) * | 2020-10-15 | 2021-01-26 | 北京大学 | 特异性表达hMRGPRX4的转基因大鼠的构建方法及其应用 |
CN112608939A (zh) * | 2020-11-27 | 2021-04-06 | 复旦大学附属肿瘤医院 | 一种条件性mTERT过表达的小鼠模型的构建方法及其应用 |
CN113412820A (zh) * | 2021-06-22 | 2021-09-21 | 中国检验检疫科学研究院 | 一种多器官细胞基因突变检测基因编辑小鼠模型的构建及应用 |
CN113789348A (zh) * | 2021-11-17 | 2021-12-14 | 北京大学第三医院(北京大学第三临床医学院) | 一种apex2基因敲入的小鼠动物模型、构建方法及其应用 |
-
2019
- 2019-11-15 CN CN201911120817.1A patent/CN110771573B/zh not_active Expired - Fee Related
Cited By (7)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN111500639A (zh) * | 2020-04-15 | 2020-08-07 | 徐州医科大学 | Stat3线粒体定位条件性基因敲入小鼠模型的构建方法 |
CN111718932A (zh) * | 2020-06-08 | 2020-09-29 | 中国人民解放军军事科学院军事医学研究院 | 一种新型的基因编辑动物生物反应器制备方法及应用 |
CN112266931A (zh) * | 2020-10-15 | 2021-01-26 | 北京大学 | 特异性表达hMRGPRX4的转基因大鼠的构建方法及其应用 |
CN112266931B (zh) * | 2020-10-15 | 2022-04-29 | 海湃泰克(北京)生物医药科技有限公司 | 特异性表达hMRGPRX4的转基因大鼠的构建方法及其应用 |
CN112608939A (zh) * | 2020-11-27 | 2021-04-06 | 复旦大学附属肿瘤医院 | 一种条件性mTERT过表达的小鼠模型的构建方法及其应用 |
CN113412820A (zh) * | 2021-06-22 | 2021-09-21 | 中国检验检疫科学研究院 | 一种多器官细胞基因突变检测基因编辑小鼠模型的构建及应用 |
CN113789348A (zh) * | 2021-11-17 | 2021-12-14 | 北京大学第三医院(北京大学第三临床医学院) | 一种apex2基因敲入的小鼠动物模型、构建方法及其应用 |
Also Published As
Publication number | Publication date |
---|---|
CN110771573B (zh) | 2021-08-27 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
CN110771573B (zh) | PirB基因敲入的小鼠动物模型及其构建方法 | |
US11317611B2 (en) | Genetically modified non-human animal with human or chimeric PD-L1 | |
US11279948B2 (en) | Genetically modified non-human animal with human or chimeric OX40 | |
US11071290B2 (en) | Genetically modified non-human animal with human or chimeric CTLA-4 | |
WO2018041121A1 (en) | Genetically modified non-human animal with human or chimeric ctla-4 | |
US10945418B2 (en) | Genetically modified non-human animal with human or chimeric PD-L1 | |
WO2018068756A1 (en) | Genetically modified non-human animal with human or chimeric btla | |
WO2018041120A1 (en) | Genetically modified non-human animal with human or chimeric tigit | |
WO2018086583A1 (en) | Genetically modified non-human animal with human or chimeric lag-3 | |
US20190373866A1 (en) | Genetically Modified Non-Human Animal With Human Or Chimeric OX40 | |
CN110804629A (zh) | PirB基因敲除小鼠动物模型及其构建方法 | |
US20190373868A1 (en) | Genetically modified non-human animal with human or chimeric pd-1 | |
EP3507376A1 (en) | Genetically modified non-human animal with human or chimeric tigit | |
CN111690689B (zh) | 人源化ccr2基因改造动物模型的构建方法及其应用 | |
CN110438160B (zh) | 一种Cd2ap基因敲除动物的构建方法及应用 | |
CN113373178A (zh) | 一种tlr8基因人源化动物模型的构建方法及应用 | |
WO2021254491A1 (en) | Genetically modified non-human animal expressing a b2m/fcrn fusion protein | |
CN113897369A (zh) | KRT10定点基因敲入P2A-CrePR1-T2A-tdTomato小鼠模型的构建及应用 | |
CN106755024B (zh) | 猪LepR基因敲除打靶载体、其构建方法及应用 | |
CN114410630B (zh) | 一种tbc1d8b基因敲除小鼠动物模型的构建方法及其应用 | |
CN116083482A (zh) | B细胞条件性敲除dhx9基因小鼠模型的构建方法 | |
CN115074384A (zh) | 一种可识别核微丝结构的nAC荧光探针小鼠模型及其应用 | |
CN114107382A (zh) | G3bp1条件性基因敲除小鼠模型 | |
CN109694885B (zh) | 基于CRISPR/Cas9技术制备PI3Kγ全身敲除模式小鼠方法及其应用和试剂盒 | |
CN113999873B (zh) | 一种基因修饰的非人动物的构建方法及其应用 |
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 | ||
CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20210827 |
|
CF01 | Termination of patent right due to non-payment of annual fee |