CN114958917A - 一种克罗恩病小鼠动物模型的构建方法和应用 - Google Patents
一种克罗恩病小鼠动物模型的构建方法和应用 Download PDFInfo
- Publication number
- CN114958917A CN114958917A CN202210704359.1A CN202210704359A CN114958917A CN 114958917 A CN114958917 A CN 114958917A CN 202210704359 A CN202210704359 A CN 202210704359A CN 114958917 A CN114958917 A CN 114958917A
- Authority
- CN
- China
- Prior art keywords
- mouse
- disease
- crohn
- knockout
- animal model
- 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.)
- Pending
Links
- 241000699666 Mus <mouse, genus> Species 0.000 title claims abstract description 97
- 208000011231 Crohn disease Diseases 0.000 title claims abstract description 45
- 238000010171 animal model Methods 0.000 title claims abstract description 29
- 238000010276 construction Methods 0.000 title claims abstract description 17
- 101150083031 Nod2 gene Proteins 0.000 claims abstract description 32
- 238000000034 method Methods 0.000 claims abstract description 32
- 108091026890 Coding region Proteins 0.000 claims abstract description 11
- 238000003209 gene knockout Methods 0.000 claims abstract description 10
- 101100080186 Mus musculus Nod2 gene Proteins 0.000 claims abstract description 8
- 230000037433 frameshift Effects 0.000 claims abstract description 6
- 108020004414 DNA Proteins 0.000 claims description 13
- 230000003321 amplification Effects 0.000 claims description 13
- 238000003199 nucleic acid amplification method Methods 0.000 claims description 13
- 238000011813 knockout mouse model Methods 0.000 claims description 9
- 210000004681 ovum Anatomy 0.000 claims description 9
- 238000012163 sequencing technique Methods 0.000 claims description 7
- 241000581650 Ivesia Species 0.000 claims description 6
- 241000699670 Mus sp. Species 0.000 claims description 6
- 210000000805 cytoplasm Anatomy 0.000 claims description 6
- 210000003101 oviduct Anatomy 0.000 claims description 6
- 238000012408 PCR amplification Methods 0.000 claims description 5
- 238000000520 microinjection Methods 0.000 claims description 5
- 238000002347 injection Methods 0.000 claims description 4
- 239000007924 injection Substances 0.000 claims description 4
- 102000011022 Chorionic Gonadotropin Human genes 0.000 claims description 3
- 108010062540 Chorionic Gonadotropin Proteins 0.000 claims description 3
- 102000002322 Egg Proteins Human genes 0.000 claims description 3
- 108010000912 Egg Proteins Proteins 0.000 claims description 3
- 102000006771 Gonadotropins Human genes 0.000 claims description 3
- 108010086677 Gonadotropins Proteins 0.000 claims description 3
- 210000000683 abdominal cavity Anatomy 0.000 claims description 3
- 239000003708 ampul Substances 0.000 claims description 3
- 210000002459 blastocyst Anatomy 0.000 claims description 3
- 239000003814 drug Substances 0.000 claims description 3
- 239000002622 gonadotropin Substances 0.000 claims description 3
- 239000001963 growth medium Substances 0.000 claims description 3
- 210000001161 mammalian embryo Anatomy 0.000 claims description 3
- 230000013011 mating Effects 0.000 claims description 3
- 244000052769 pathogen Species 0.000 claims description 3
- 230000001717 pathogenic effect Effects 0.000 claims description 3
- 230000001850 reproductive effect Effects 0.000 claims description 3
- 210000002966 serum Anatomy 0.000 claims description 3
- 238000001356 surgical procedure Methods 0.000 claims description 3
- 238000005406 washing Methods 0.000 claims description 3
- 241000699694 Gerbillinae Species 0.000 claims 1
- 229940084986 human chorionic gonadotropin Drugs 0.000 claims 1
- 238000004519 manufacturing process Methods 0.000 claims 1
- 239000000203 mixture Substances 0.000 claims 1
- 230000002028 premature Effects 0.000 claims 1
- 201000010099 disease Diseases 0.000 abstract description 14
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 abstract description 14
- 108090000623 proteins and genes Proteins 0.000 abstract description 12
- 108091033409 CRISPR Proteins 0.000 abstract description 10
- 238000010172 mouse model Methods 0.000 abstract description 6
- 231100000221 frame shift mutation induction Toxicity 0.000 abstract description 5
- 238000010354 CRISPR gene editing Methods 0.000 abstract description 4
- 238000010356 CRISPR-Cas9 genome editing Methods 0.000 abstract description 3
- 230000003950 pathogenic mechanism Effects 0.000 abstract description 3
- 229940126585 therapeutic drug Drugs 0.000 abstract 1
- 108020005004 Guide RNA Proteins 0.000 description 11
- 238000005516 engineering process Methods 0.000 description 7
- 101001125026 Homo sapiens Nucleotide-binding oligomerization domain-containing protein 2 Proteins 0.000 description 6
- 102100029441 Nucleotide-binding oligomerization domain-containing protein 2 Human genes 0.000 description 6
- 238000006243 chemical reaction Methods 0.000 description 5
- 206010009944 Colon cancer Diseases 0.000 description 4
- 238000010362 genome editing Methods 0.000 description 4
- 230000035772 mutation Effects 0.000 description 4
- 230000008506 pathogenesis Effects 0.000 description 4
- 238000010459 TALEN Methods 0.000 description 3
- 108010043645 Transcription Activator-Like Effector Nucleases Proteins 0.000 description 3
- 208000029742 colonic neoplasm Diseases 0.000 description 3
- 238000011161 development Methods 0.000 description 3
- 230000002068 genetic effect Effects 0.000 description 3
- 230000003834 intracellular effect Effects 0.000 description 3
- 206010061218 Inflammation Diseases 0.000 description 2
- 108700026244 Open Reading Frames Proteins 0.000 description 2
- 230000009471 action Effects 0.000 description 2
- 101150038500 cas9 gene Proteins 0.000 description 2
- 238000010586 diagram Methods 0.000 description 2
- 235000013601 eggs Nutrition 0.000 description 2
- 210000001035 gastrointestinal tract Anatomy 0.000 description 2
- 230000007614 genetic variation Effects 0.000 description 2
- 238000012268 genome sequencing Methods 0.000 description 2
- 210000003405 ileum Anatomy 0.000 description 2
- 230000004054 inflammatory process Effects 0.000 description 2
- 101150017145 nod gene Proteins 0.000 description 2
- 102000054765 polymorphisms of proteins Human genes 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 238000012216 screening Methods 0.000 description 2
- 230000008685 targeting Effects 0.000 description 2
- 208000036652 Adenocarcinoma of the small intestine Diseases 0.000 description 1
- 206010008609 Cholangitis sclerosing Diseases 0.000 description 1
- 108020004705 Codon Proteins 0.000 description 1
- 208000001333 Colorectal Neoplasms Diseases 0.000 description 1
- 208000036649 Dysbacteriosis Diseases 0.000 description 1
- 208000027244 Dysbiosis Diseases 0.000 description 1
- 108700024394 Exon Proteins 0.000 description 1
- 206010016654 Fibrosis Diseases 0.000 description 1
- 208000017604 Hodgkin disease Diseases 0.000 description 1
- 208000021519 Hodgkin lymphoma Diseases 0.000 description 1
- 208000010747 Hodgkins lymphoma Diseases 0.000 description 1
- 206010021307 Ileal stenosis Diseases 0.000 description 1
- 208000022559 Inflammatory bowel disease Diseases 0.000 description 1
- 102000043136 MAP kinase family Human genes 0.000 description 1
- 108091054455 MAP kinase family Proteins 0.000 description 1
- 208000015914 Non-Hodgkin lymphomas Diseases 0.000 description 1
- 108010079933 Receptor-Interacting Protein Serine-Threonine Kinase 2 Proteins 0.000 description 1
- 102100022502 Receptor-interacting serine/threonine-protein kinase 2 Human genes 0.000 description 1
- 102100030571 STE20-like serine/threonine-protein kinase Human genes 0.000 description 1
- 101710157230 STE20-like serine/threonine-protein kinase Proteins 0.000 description 1
- 101710202841 Serine/threonine-protein kinase 2 Proteins 0.000 description 1
- 101710091457 Serine/threonine-protein kinase Nek4 Proteins 0.000 description 1
- 108091081024 Start codon Proteins 0.000 description 1
- 210000001744 T-lymphocyte Anatomy 0.000 description 1
- 108010006785 Taq Polymerase Proteins 0.000 description 1
- 238000000246 agarose gel electrophoresis Methods 0.000 description 1
- 230000000844 anti-bacterial effect Effects 0.000 description 1
- 210000003719 b-lymphocyte Anatomy 0.000 description 1
- 230000001580 bacterial effect Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 210000004027 cell Anatomy 0.000 description 1
- 230000030833 cell death Effects 0.000 description 1
- 208000006990 cholangiocarcinoma Diseases 0.000 description 1
- 210000000349 chromosome Anatomy 0.000 description 1
- 230000001684 chronic effect Effects 0.000 description 1
- 210000001072 colon Anatomy 0.000 description 1
- 238000005336 cracking Methods 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
- 238000005520 cutting process Methods 0.000 description 1
- 238000012217 deletion Methods 0.000 description 1
- 230000037430 deletion Effects 0.000 description 1
- 210000004443 dendritic cell Anatomy 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 230000007140 dysbiosis Effects 0.000 description 1
- 238000001962 electrophoresis Methods 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 230000004761 fibrosis Effects 0.000 description 1
- 238000012239 gene modification Methods 0.000 description 1
- 244000005709 gut microbiome Species 0.000 description 1
- 210000003958 hematopoietic stem cell Anatomy 0.000 description 1
- 210000003630 histaminocyte Anatomy 0.000 description 1
- 201000002316 ileum cancer Diseases 0.000 description 1
- 230000007124 immune defense Effects 0.000 description 1
- 230000028993 immune response Effects 0.000 description 1
- 230000002401 inhibitory effect Effects 0.000 description 1
- 238000007689 inspection Methods 0.000 description 1
- 210000002490 intestinal epithelial cell Anatomy 0.000 description 1
- 239000003446 ligand Substances 0.000 description 1
- 210000002540 macrophage Anatomy 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 230000000813 microbial effect Effects 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 238000010369 molecular cloning Methods 0.000 description 1
- 239000013642 negative control Substances 0.000 description 1
- 210000004967 non-hematopoietic stem cell Anatomy 0.000 description 1
- 239000002773 nucleotide Substances 0.000 description 1
- 125000003729 nucleotide group Chemical group 0.000 description 1
- 230000037361 pathway Effects 0.000 description 1
- 102000007863 pattern recognition receptors Human genes 0.000 description 1
- 108010089193 pattern recognition receptors Proteins 0.000 description 1
- 239000013612 plasmid Substances 0.000 description 1
- 108091033319 polynucleotide Proteins 0.000 description 1
- 239000002157 polynucleotide Substances 0.000 description 1
- 102000040430 polynucleotide Human genes 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 201000000742 primary sclerosing cholangitis Diseases 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 230000000770 proinflammatory effect Effects 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 230000008844 regulatory mechanism Effects 0.000 description 1
- 230000004044 response Effects 0.000 description 1
- 208000010157 sclerosing cholangitis Diseases 0.000 description 1
- 230000035945 sensitivity Effects 0.000 description 1
- 238000012772 sequence design Methods 0.000 description 1
- 230000019491 signal transduction Effects 0.000 description 1
- 206010073373 small intestine adenocarcinoma Diseases 0.000 description 1
- 206010041823 squamous cell carcinoma Diseases 0.000 description 1
- 210000000130 stem cell Anatomy 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 238000002560 therapeutic procedure Methods 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
- 238000005303 weighing Methods 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
- A01K67/0276—Knock-out vertebrates
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K49/00—Preparations for testing in vivo
- A61K49/0004—Screening or testing of compounds for diagnosis of disorders, assessment of conditions, e.g. renal clearance, gastric emptying, testing for diabetes, allergy, rheuma, pancreas functions
- A61K49/0008—Screening agents using (non-human) animal models or transgenic animal models or chimeric hosts, e.g. Alzheimer disease animal model, transgenic model for heart failure
-
- C—CHEMISTRY; METALLURGY
- 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
- C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
- C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
- C12N15/1138—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against receptors or cell surface proteins
-
- 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
- C12N15/89—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation using microinjection
-
- 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
-
- 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
- C12N2310/00—Structure or type of the nucleic acid
- C12N2310/10—Type of nucleic acid
- C12N2310/20—Type of nucleic acid involving clustered regularly interspaced short palindromic repeats [CRISPRs]
-
- 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
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Genetics & Genomics (AREA)
- Engineering & Computer Science (AREA)
- Zoology (AREA)
- Biomedical Technology (AREA)
- Biotechnology (AREA)
- Chemical & Material Sciences (AREA)
- General Engineering & Computer Science (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Wood Science & Technology (AREA)
- Organic Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Molecular Biology (AREA)
- Biophysics (AREA)
- Plant Pathology (AREA)
- Microbiology (AREA)
- Physics & Mathematics (AREA)
- Veterinary Medicine (AREA)
- Biochemistry (AREA)
- Animal Behavior & Ethology (AREA)
- Environmental Sciences (AREA)
- Pathology (AREA)
- Biodiversity & Conservation Biology (AREA)
- Diabetes (AREA)
- Endocrinology (AREA)
- Gastroenterology & Hepatology (AREA)
- Animal Husbandry (AREA)
- Rheumatology (AREA)
- Toxicology (AREA)
- Urology & Nephrology (AREA)
- Epidemiology (AREA)
- Public Health (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
本发明属于转基因技术领域,具体地涉及一种克罗恩病小鼠动物模型的构建方法和应用,所述方法包括使用CRISPR‑Cas9靶向敲除小鼠Nod2基因;具体的,所述敲除小鼠Nod2基因,为敲除Nod2基因Exon 3~4之间的序列;敲除的序列占Nod2基因整个编码区的63.24%,所敲区域序列为非3倍数,导致移码突变,造成编码区域提前终止;包括以下步骤:合成gRNA1=SEQ ID NO:1=TCTGTCGTTAGACGTGGGTCGGG;合成gRNA2=SEQ ID NO:2=TGGATGCGTTTCCACTGCTCTGG;本发明使用CRISPR/Cas9系统构建Nod2基因敲除小鼠模型方法,步骤简单易行,周期短,拿到阳性小鼠的概率高,利用该小鼠模型可以加快研究该疾病的致病机理的进度,并为进一步开发针对该疾病的治疗药物提供服务。
Description
技术领域
本发明属于转基因技术领域,具体地涉及一种克罗恩病小鼠动物模型的构建方法和应用。
背景技术
克罗恩病(Crohn’s disease,CD)是一种慢性炎症性肠病,又称局限性肠炎、局限性回肠炎、节段性肠炎和肉芽肿性肠炎。此病可以影响肠道的任何部位,但好发于末端回肠和右半结肠,可能会引起炎症甚至纤维化,导致回肠管壁狭窄。此病易反复发作,目前尚不能治愈。患有克罗恩疾病的人群与普通人群相比较回肠和大肠癌发生率高出90~100倍,克罗恩疾病患者出现阴肛鳞癌、霍奇金病和非霍奇金淋巴瘤等疾病也比较多,小肠部癌变腺癌是最常见的类型,多见于年轻人,患者累及结肠者会增加罹患结肠癌的风险,伴有原发性硬化性胆管炎者会增加患胆管癌和结肠癌的风险。目前克罗恩疾病发病机制尚不十分明确,但资料显示NOD2的基因突变是导致该疾病发生的危险因素。
随着分子生物学和遗传学研究的深入,NOD2基因与克罗恩病易患体质的关系逐渐明确。NOD2基因的遗传变异是克罗恩病最强烈的遗传风险关联,大约 20%的患病风险与该基因的三种多核苷酸多态性有关。NOD2是一种细胞内模式识别受体,由多种细胞表达,包括造血细胞(如T细胞、B细胞、巨噬细胞、树突状细胞核肥大细胞)和非造血细胞(如潘氏细胞、干细胞、杯状细胞和肠上皮细胞),可以识别病原相关的肽聚糖并触发一系列的促炎和抗菌免疫反应,在细胞内微生物的免疫防御系统中具有重要作用。NOD2在胞浆中处于一种抑制的单体状态,配体识别时激活构象,招募丝氨酸/苏氨酸蛋白激酶2(RIPK2),激活 IKK复合物和MAPK途径,调控NF-κB信号通路。NOD基因功能的缺失影响肠道微生物群的黏膜反应的调节机制,这与克罗恩病肠道炎症和菌群失调有关。为了研究该疾病的发病机制以及开发有效的治疗方法,构建该疾病的实验模型对于疾病的研究不可或缺。
传统的ZNF和TALEN编辑技术分别有各自的缺点,比如ZNF技术易于脱靶,导致细胞死亡或者额外突变。TALEN运用起来更简单、构建更便捷,且价格更低廉。但是装配TALEN编码质粒却是一个冗长的、高强度工作的过程。因此,急需的寻找更新的技术来对NOD基因进行基因编辑,构建新型的克罗恩病小鼠动物模型。
发明内容
为了解决上述技术问题,发明人公开了一种克罗恩病小鼠动物模型的构建方法和应用。
本发明的技术方案如下:
一种克罗恩病小鼠动物模型的构建方法,所述方法包括使用CRISPR-Cas9 靶向敲除小鼠Nod2基因。
使用CRISPR-Cas9技术,是继ZFN、TALENs等基因编辑技术推出后的第三代基因编辑技术,成为现有基因编辑和基因修饰里面效率最高、最简便、成本最低、最容易上手的技术之一,但是目前还未有CRISPR-Cas9定向敲除小鼠Nod2 基因的报道,本发明具有开拓性。
进一步的,上述一种克罗恩病小鼠动物模型的构建方法,所述敲除小鼠Nod2 基因,为敲除Nod2基因Exon 3~4之间的序列。Nod2基因位于小鼠8号染色体上,共有4种转录本形式,我们选择Nod2-203作为目标转录本,该转录本共有 13个外显子(Exon),起始密码子ATG位于Exon 2,终止密码子TGA位于Exon 12。
进一步的,上述一种克罗恩病小鼠动物模型的构建方法,敲除的序列占Nod2 基因整个编码区的63.24%,所敲区域序列为非3倍数,导致移码突变,造成编码区域提前终止。本发明共设计两条gRNA,gRNA1靶向切割Exon3的5’端, gRNA2靶向切割Exon 4的3’端,这两条gRNA在Cas9蛋白的作用下可以切除 Exon 3~4之间的序列,敲除的Exon 3起始于整个编码区域的15.14%,敲除的 Exon 3~4占据整个编码区域的63.24%(且该敲除区域不含有其他已知基因),敲除区域非3的倍数,导致移码突变造成编码区域提前终止,从而达到敲除Nod2 的目的。
进一步的,上述一种克罗恩病小鼠动物模型的构建方法,包括以下步骤:合成gRNA1=SEQ ID NO:1=TCTGTCGTTAGACGTGGGTCGGG;合成gRNA2= SEQ ID NO:2=TGGATGCGTTTCCACTGCTCTGG。经过测试,上述gRNA能够精确的引导Cas9蛋白进行精确切割,脱靶几率小,成功率高。
进一步的,上述一种克罗恩病小鼠动物模型的构建方法,包括以下步骤:
1)原核注射:将Cas9mRNA与人工合成的针对Nod2基因敲除的gRNA1 和gRNA2混匀后通过显微注射至小鼠受精卵胞质或原核中,得F0小鼠;
2)Nod2基因敲除小鼠的PCR鉴定:将F0小鼠进行PCR鉴定,并与野生型小鼠交配获得F1小鼠,鉴定获得克罗恩病小鼠动物模型。
上述构建方法,成功率高,约3~5个月可获得克罗恩小鼠动物模型,相比现有技术时间大大缩短。
进一步的,上述一种克罗恩病小鼠动物模型的构建方法,所述步骤1)包括以下步骤:
选取4~6周龄SPF级雌性小鼠作为卵子供体,小鼠腹腔注射PMSG(孕马血清促性腺素),48h后注射hCG(人血绒膜促性腺素),随即与生殖能力正常的种公鼠进行交配,从输卵管收集小鼠受精卵,消化洗涤之后置于37℃培养箱中待用;将Cas9mRNA与人工合成的针对Nod2基因敲除的gRNA1和gRNA2 混匀后通过显微注射至小鼠受精卵胞质或原核中,注射后的胚胎保存在特定培养基中,37℃,5%CO2温箱中培养至3.5天,每15~30个囊胚移植到代孕雌鼠的输卵管壶腹部中,代孕鼠每隔一周进行体重称量,初步判断是否怀孕,手术后 19~21天仔鼠分娩,待仔鼠5天后剪鼠尾编号送检。
上述注射方法技术成熟,成功率高,筛选方便。
进一步的,上述一种克罗恩病小鼠动物模型的构建方法,所述步骤2)包括以下步骤:
根据Nod2基因敲除区域,分别在Nod2基因的Exon 3的5’端以及Exon 4 的3’端设计一对引物F1R1,对出生的F0小鼠鼠尾提取DNA,进行PCR扩增并测序,将鉴定正确的阳性小鼠与野生型小鼠交配获得F1小鼠,小鼠出生14天后进行PCR扩增鉴定小鼠纯杂合,鉴定引物为一对F1R1和一对F2R1;纯合小鼠扩增产物为456bp,杂合小鼠扩增产物为456bp/574bp,野生型小鼠扩增产物为574bp。
上述鉴定方法步骤少,简单,精确度高。
进一步的,上述一种克罗恩病小鼠动物模型的构建方法,所述一对引物F1R1 分别为:F1=SEQ ID NO:3=AGGTTGGCTGTGTGTTTGTTTCAC;R1=SEQ ID NO:4=CAGACATGGAAAGAGGCCCACAG。
进一步的,上述一种克罗恩病小鼠动物模型的构建方法,所述一对引物F2R1 分别为:F2=SEQ ID NO:5=TCAAGGAAATGACCCATAGACAAGC;R1=SEQ ID NO:4=CAGACATGGAAAGAGGCCCACAG。
上述两对引物进行鉴定,特异性好,灵敏度高。
进一步的,上述一种克罗恩病小鼠动物模型的构建方法在制备治疗克罗恩病的药物中的应用。目前克罗恩疾病发病机制尚不十分明确,但资料显示NOD2 的基因突变是导致该疾病发生的危险因素。NOD2基因的遗传变异是克罗恩病最强的遗传风险;大约20%的患病风险与该基因的三个单核苷酸多态性有关。此外, NOD2突变是回肠狭窄、需要手术的强预测因子。因此构建NOD2敲除的小鼠模型,对于研制NOD2基因相关疾病的药物具有重大帮助。
本发明具有以下有益效果:
本发明利用CRISPR/Cas9技术构建Nod2基因敲除小鼠模型,Nod2作为细胞内细菌识别分子的功能将被进一步揭示,一些与Nod2相关的因子及其作用机制也将被逐步发现,进而揭示这一功能系统的全貌,为克罗恩疾病发病机制的深入研究。本发明设计了特异性靶向Nod2基因的两条gRNA,利用cas9蛋白敲除 Nod2基因的Exon 3~4,所敲区域序列为非3倍数,导致移码突变,造成编码区域提前终止,从而达到敲除基因的目的。使用CRISPR/Cas9系统构建Nod2基因敲除小鼠模型方法步骤简单易行,周期短,拿到阳性小鼠的概率高,利用该小鼠模型可以加快研究该疾病的致病机理的进度,并为进一步开发针对该疾病的治疗方式提供服务。
附图说明
附图1Nod2基因敲除小鼠的构建方案示意图;图中深色区域表示敲除区域,包裹实心矩形区域代表基因的外显子,gRNA region代表gRNA的剪切区域;F1,R1代表用于小鼠鉴定的引物结合区域;
附图2为Nod2基因敲除小鼠的鉴定策略示意图;图中深色区域表示敲除区域,包裹实心矩形区域代表基因的外显子,gRNA region代表gRNA的剪切区域; F1,F2,R1代表用于小鼠鉴定的引物结合区域;
附图3为Nod2基因敲除小鼠的鉴定结果;利用PCR引物PCR Primers 1进行PCR筛选,基因敲除后的扩增产物:456bp;野生型的扩增产物:6469bp;左图为DNA分子量标记,右图为PCR的鉴定产物图。PCR反应在25μL体系中扩增35个循环,Taq DNA聚合酶使用P222,使用的阴性对照为:Water(不加DNA模板)和WT(小鼠基因组DNA);
附图4为Nod2基因敲除小鼠的基因组测序结果;箭头处指示Nod2基因被删除6013bp;测序引物序列为SEQ ID NO: 6=5’-AGGTTGGCTGTGTGTTTGTTTCAC-3’。
具体实施方式
下面结合实例对本发明的方法做进一步说明,实施例中未注明具体条件的实验方法,通常可按常规条件,如J.萨姆布鲁克(Sambrook)等编写的《分子克隆实验指南》中所述的条件,或按照制造厂商所建议的条件进行。本领域相关的技术人员可以借助实施例更好地理解和掌握本发明。但是,实现本发明的方法不应限于本发明实施例所记载的具体方法步骤。
实施例1
敲除方案设计:
根据需求,查找Nod2基因详细信息,选择基因敲除的区域需尽可能包括基因的功能域部分,最好占据编码区的前50%,另外也要根据实际情况选择敲除区域,我们选择敲除Nod2基因Exon 3~4,包含Nod2基因整个编码区的63.24%,如附图1所示。
实施例2
gRNA序列设计
根据小鼠Nod2基因的序列,设计并合成了两条针对该基因的gRNA序列,序列信息。
合成gRNA1=SEQ ID NO:1=TCTGTCGTTAGACGTGGGTCGGG;
合成gRNA2=SEQ ID NO:2=TGGATGCGTTTCCACTGCTCTGG。
实施例3
原核注射
选取4~6周龄SPF级雌性小鼠(Cyagen)作为卵子供体,小鼠腹腔注射PMSG (孕马血清促性腺素),48h后注射hCG(人血绒膜促性腺素),随即与生殖能力正常的种公鼠(Cyagen)进行交配,从输卵管收集小鼠受精卵,消化洗涤之后置于37℃培养箱中待用。将Cas9mRNA(20~200ng/μL)与人工合成的针对Nod2 基因敲除的gRNA1和gRNA2(20~50ng/μL)混匀后通过显微注射至小鼠受精卵胞质或原核中,注射后的胚胎保存在特定培养基中,37℃,5%CO2温箱中培养至3.5天,每15~30个囊胚移植到代孕雌鼠的输卵管壶腹部中,代孕鼠每隔一周进行体重称量,初步判断是否怀孕,手术后19~21天仔鼠分娩,待仔鼠5天后剪鼠尾编号送检。
实施例4
Nod2基因敲除小鼠的PCR鉴定
鉴定策略示意图见附图2,根据Nod2基因敲除区域,我们分别在Exon 3的 5’端以及Exon 4的3’端设计一对引物F1R1(F1=SEQ ID NO:3=5’-AGGTTGGCTGTGTGTTTGTTTCAC-3’;R1=SEQ ID NO: 4=5’-CAGACATGGAAAGAGGCCCACAG-3’),对出生的F0小鼠鼠尾提取DNA,进行PCR扩增并测序,该引物在基因敲除小鼠中扩增的产物为456bp,在野生型小鼠中以相同的程序不能扩增出产物,鉴定结果及测序结果如附图3和4所示。将鉴定正确的阳性小鼠与野生型小鼠交配获得F1小鼠,小鼠出生14天后进行 PCR扩增鉴定小鼠纯杂合,鉴定引物如附图2所示,使用两对引物F1R1(F1=SEQ ID NO:3=5’-AGGTTGGCTGTGTGTTTGTTTCAC-3’;R1=SEQ ID NO: 4=5’-CAGACATGGAAAGAGGCCCACAG-3’)和F2R1(F2=SEQ ID NO: 5=5’-TCAAGGAAATGACCCATAGACAAGC-3’;R1=SEQ ID NO: 4=5’-CAGACATGGAAAGAGGCCCACAG-3’),纯合小鼠扩增产物为456bp,杂合小鼠扩增产物为456bp/574bp,野生型小鼠扩增产物为574bp。
小鼠PCR鉴定部位为鼠尾,对于鼠尾鉴定选择粗裂解的方法提取DNA。
PCR反应体系如下表1所示
表1 PCR反应体系
PCR反应程序如下表2所示:
表2PCR反应程序
将PCR产物进行琼脂糖凝胶电泳,电泳结果如附图3所示。
实施例5
基因组测序
对Nod2基因敲除小鼠的基因组测序,测序结果见附图4,箭头处指示Nod2 基因被删除6013bp,证明敲除成功,测序引物为SEQ ID NO: 6=5’-AGGTTGGCTGTGTGTTTGTTTCAC-3’。
由以上实施例可知:本发明设计了特异性靶向Nod2基因的两条gRNA,利用cas9蛋白敲除Nod2基因的Exon 3~4,所敲区域序列为非3倍数,导致移码突变,造成编码区域提前终止,从而达到敲除基因的目的。使用CRISPR/Cas9 系统构建Nod2基因敲除小鼠模型方法步骤简单易行,周期短,拿到阳性小鼠的概率高,利用该小鼠模型可以加快研究该疾病的致病机理的进度,并为进一步开发针对该疾病的治疗方式提供服务。相比传统的构建方法,本发明能更简单、高效的对基因进行修饰。
以上仅为本发明的较佳实施例而已,不能以此限定本发明的保护范围,即大凡依本发明权利要求书及发明内容所做的简单的等效变化与修改,皆仍属于本发明专利申请的保护范围。
SEQUENCE LISTING
<110> 赛业(苏州)生物科技有限公司
<120> 一种克罗恩病小鼠动物模型的构建方法和应用
<130> 2022
<160> 6
<170> PatentIn version 3.5
<210> 1
<211> 23
<212> DNA
<213> artificial
<400> 1
tctgtcgtta gacgtgggtc ggg 23
<210> 2
<211> 23
<212> DNA
<213> artificial
<400> 2
tggatgcgtt tccactgctc tgg 23
<210> 3
<211> 24
<212> DNA
<213> artificial
<400> 3
aggttggctg tgtgtttgtt tcac 24
<210> 4
<211> 23
<212> DNA
<213> artificial
<400> 4
cagacatgga aagaggccca cag 23
<210> 5
<211> 25
<212> DNA
<213> artificial
<400> 5
tcaaggaaat gacccataga caagc 25
<210> 6
<211> 24
<212> DNA
<213> artificial
<400> 6
aggttggctg tgtgtttgtt tcac 24
Claims (10)
1.一种克罗恩病小鼠动物模型的构建方法,其特征在于,所述方法包括使用CRISPR-Cas9靶向敲除小鼠Nod2基因。
2.根据权利要求1所述的一种克罗恩病小鼠动物模型的构建方法,其特征在于,所述敲除小鼠Nod2基因,为敲除Nod2基因Exon 3~4之间的序列。
3.根据权利要求2所述的一种克罗恩病小鼠动物模型的构建方法,其特征在于,敲除的序列占Nod2基因整个编码区的63.24%,所敲区域序列为非3倍数,导致移码突变,造成编码区域提前终止。
4.根据权利要求3所述的一种克罗恩病小鼠动物模型的构建方法,其特征在于,包括以下步骤:合成gRNA1=SEQ ID NO:1= TCTGTCGTTAGACGTGGGTCGGG;合成gRNA2= SEQ ID NO:2=TGGATGCGTTTCCACTGCTCTGG。
5.根据权利要求4所述的一种克罗恩病小鼠动物模型的构建方法,其特征在于,包括以下步骤:
1)原核注射:将Cas9 mRNA与人工合成的针对Nod2基因敲除的gRNA1和gRNA2混匀后通过显微注射至小鼠受精卵胞质或原核中,得F0小鼠;
2)Nod2基因敲除小鼠的PCR鉴定:将F0小鼠进行PCR鉴定,并与野生型小鼠交配获得F1小鼠,鉴定获得克罗恩病小鼠动物模型。
6.根据权利要求5所述的一种克罗恩病小鼠动物模型的构建方法,其特征在于,所述步骤1)包括以下步骤:
选取4~6周龄SPF级雌性小鼠作为卵子供体,小鼠腹腔注射PMSG孕马血清促性腺素,48h后注射hCG人血绒膜促性腺素,随即与生殖能力正常的种公鼠进行交配,从输卵管收集小鼠受精卵,消化洗涤之后置于37℃培养箱中待用;将Cas9 mRNA与人工合成的针对Nod2基因敲除的gRNA1和gRNA2混匀后通过显微注射至小鼠受精卵胞质或原核中,注射后的胚胎保存在特定培养基中,37℃,5% CO2温箱中培养至3.5天,每15~30个囊胚移植到代孕雌鼠的输卵管壶腹部中,代孕鼠每隔一周进行体重称量,初步判断是否怀孕,手术后19~21天仔鼠分娩,待仔鼠5天后剪鼠尾编号送检。
7.根据权利要求5所述的一种克罗恩病小鼠动物模型的构建方法,其特征在于,所述步骤2)包括以下步骤:
根据Nod2基因敲除区域,分别在Nod2基因的Exon 3的5’端及Exon 4的3’端设计一对引物F1R1,对出生的F0小鼠鼠尾提取DNA,进行PCR扩增并测序,将鉴定正确的阳性小鼠与野生型小鼠交配获得F1小鼠,小鼠出生14天后进行PCR扩增鉴定小鼠纯杂合,鉴定引物为一对F1R1和一对F2R1;纯合小鼠扩增产物为456 bp,杂合小鼠扩增产物为456 bp/574 bp,野生型小鼠扩增产物为574 bp。
8.根据权利要求7所述的一种克罗恩病小鼠动物模型的构建方法,其特征在于,所述一对引物F1R1分别为:F1=SEQ ID NO:3 =AGGTTGGCTGTGTGTTTGTTTCAC;R1=SEQ ID NO:4=CAGACATGGAAAGAGGCCCACAG。
9.根据权利要求7所述的一种克罗恩病小鼠动物模型的构建方法,其特征在于,所述一对引物F2R1分别为:F2=SEQ ID NO:5= TCAAGGAAATGACCCATAGACAAGC;R1=SEQ ID NO:4=CAGACATGGAAAGAGGCCCACAG。
10.如权利要求1-9任一项所述的克罗恩病小鼠动物模型的构建方法在制备治疗克罗恩病的药物中的应用。
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202210704359.1A CN114958917A (zh) | 2022-06-21 | 2022-06-21 | 一种克罗恩病小鼠动物模型的构建方法和应用 |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202210704359.1A CN114958917A (zh) | 2022-06-21 | 2022-06-21 | 一种克罗恩病小鼠动物模型的构建方法和应用 |
Publications (1)
Publication Number | Publication Date |
---|---|
CN114958917A true CN114958917A (zh) | 2022-08-30 |
Family
ID=82966129
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN202210704359.1A Pending CN114958917A (zh) | 2022-06-21 | 2022-06-21 | 一种克罗恩病小鼠动物模型的构建方法和应用 |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN114958917A (zh) |
Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2021097131A1 (en) * | 2019-11-12 | 2021-05-20 | Orchard Therapeutics (Europe) Limited | Compositions and methods for treating or preventing crohn's disease |
CN113699152A (zh) * | 2021-09-15 | 2021-11-26 | 南方医科大学皮肤病医院(广东省皮肤病医院、广东省皮肤性病防治中心、中国麻风防治研究中心) | Slc35e2b基因敲除小鼠动物模型的构建方法和应用 |
CN113736787A (zh) * | 2021-09-22 | 2021-12-03 | 赛业(苏州)生物科技有限公司 | 靶向小鼠Atp7b基因的gRNA及构建Wilson疾病小鼠模型的方法 |
-
2022
- 2022-06-21 CN CN202210704359.1A patent/CN114958917A/zh active Pending
Patent Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2021097131A1 (en) * | 2019-11-12 | 2021-05-20 | Orchard Therapeutics (Europe) Limited | Compositions and methods for treating or preventing crohn's disease |
CN113699152A (zh) * | 2021-09-15 | 2021-11-26 | 南方医科大学皮肤病医院(广东省皮肤病医院、广东省皮肤性病防治中心、中国麻风防治研究中心) | Slc35e2b基因敲除小鼠动物模型的构建方法和应用 |
CN113736787A (zh) * | 2021-09-22 | 2021-12-03 | 赛业(苏州)生物科技有限公司 | 靶向小鼠Atp7b基因的gRNA及构建Wilson疾病小鼠模型的方法 |
Non-Patent Citations (3)
Title |
---|
NICOLAS ROCHEREAU等: "NOD2 deficiency increases retrograde transport of secretory IgA complexes in Crohn’s disease", NATURE COMMUNICATION, vol. 12, no. 1, pages 1 - 13 * |
SHIXUN LI等: "NOD2 negatively regulated titanium particle induced osteolysis in mice", BIOMATERIALS SCIENCE, no. 7, pages 2702 - 2715 * |
郭雪坤等: "炎症性肠病的发病机理与免疫治疗的研究进展", 生命科学, vol. 29, no. 9, pages 873 - 882 * |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
CN108660161B (zh) | 基于CRISPR/Cas9技术的制备无嵌合基因敲除动物的方法 | |
CN111926017B (zh) | 一种csf1ra基因缺失斑马鱼突变体的制备及其应用 | |
WO2020240876A1 (ja) | エクソンヒト化マウス | |
CN113755498A (zh) | 靶向小鼠Ube3a基因的gRNA及构建AS疾病小鼠模型的方法 | |
CN113736787A (zh) | 靶向小鼠Atp7b基因的gRNA及构建Wilson疾病小鼠模型的方法 | |
CN105274141A (zh) | 一种用于原始生殖细胞靶向突变的转基因载体及制备方法和用途 | |
CN110438160A (zh) | 一种Cd2ap基因敲除动物的构建方法及应用 | |
Astre et al. | The African turquoise killifish (Nothobranchius furzeri): biology and research applications | |
CN113801893A (zh) | 一种Psme3条件性基因敲除小鼠模型的构建方法及其应用 | |
CN116019063A (zh) | 一种小鼠条件性诱导Hmox1基因敲除模型的构建方法及应用 | |
CN116083492A (zh) | csde1基因缺失斑马鱼突变体的制备及斑马鱼造血干细胞发育缺陷模型的构建方法 | |
CN113957074B (zh) | 一种小脑共济失调疾病模型的构建方法及应用 | |
Kang et al. | Apancreatic pigs cloned using Pdx1-disrupted fibroblasts created via TALEN-mediated mutagenesis | |
CN104334017A (zh) | 尿激酶型纤溶酶原激活剂转基因小鼠 | |
CN110195057B (zh) | Hr基因经遗传修饰的非人动物或其子代的制备方法及应用 | |
CN114480497B (zh) | 一种ep400基因敲除斑马鱼心力衰竭模型的构建及其应用的方法 | |
CN109929876A (zh) | Vps28基因敲除小鼠动物模型的构建方法和应用 | |
CN114958917A (zh) | 一种克罗恩病小鼠动物模型的构建方法和应用 | |
CN116083482A (zh) | B细胞条件性敲除dhx9基因小鼠模型的构建方法 | |
CN115261360A (zh) | 一种gata6基因敲除斑马鱼模型的构建方法 | |
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 | |
CN114763557A (zh) | Ddx5在抗病毒和调节免疫反应中的应用 | |
Brakebusch | Generation and analysis of genetically modified mice | |
CN111849977B (zh) | 一种精子载体制备转基因动物的方法以及一种制备矮小型转基因鸡的sgRNA和制备方法 | |
CN114747541B (zh) | 一种psgl-1人源化非人类动物模型的构建方法及应用 |
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 | ||
RJ01 | Rejection of invention patent application after publication |
Application publication date: 20220830 |
|
RJ01 | Rejection of invention patent application after publication |