CN107287245B - 一种基于CRISPR/Cas9技术的Glrx1基因敲除动物模型的构建方法 - Google Patents
一种基于CRISPR/Cas9技术的Glrx1基因敲除动物模型的构建方法 Download PDFInfo
- Publication number
- CN107287245B CN107287245B CN201710390894.3A CN201710390894A CN107287245B CN 107287245 B CN107287245 B CN 107287245B CN 201710390894 A CN201710390894 A CN 201710390894A CN 107287245 B CN107287245 B CN 107287245B
- Authority
- CN
- China
- Prior art keywords
- sgrna
- glrx1
- cas9
- gene knockout
- mouse
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
- 108091033409 CRISPR Proteins 0.000 title claims abstract description 55
- 101150030440 Glrx gene Proteins 0.000 title claims abstract description 29
- 238000003209 gene knockout Methods 0.000 title claims abstract description 29
- 238000005516 engineering process Methods 0.000 title claims abstract description 19
- 238000010354 CRISPR gene editing Methods 0.000 title claims abstract description 18
- 238000010276 construction Methods 0.000 title claims abstract description 18
- 238000010171 animal model Methods 0.000 title claims abstract description 11
- 108091027544 Subgenomic mRNA Proteins 0.000 claims abstract description 44
- 235000013601 eggs Nutrition 0.000 claims abstract description 9
- 238000000034 method Methods 0.000 claims abstract description 9
- 238000013461 design Methods 0.000 claims abstract description 5
- 238000000338 in vitro Methods 0.000 claims abstract description 5
- 101000856984 Mus musculus Glutaredoxin-1 Proteins 0.000 claims abstract description 4
- 230000008685 targeting Effects 0.000 claims abstract description 4
- 241000699670 Mus sp. Species 0.000 claims description 19
- 241000699666 Mus <mouse, genus> Species 0.000 claims description 15
- 108020004999 messenger RNA Proteins 0.000 claims description 15
- 238000000137 annealing Methods 0.000 claims description 7
- 238000012163 sequencing technique Methods 0.000 claims description 7
- 239000013613 expression plasmid Substances 0.000 claims description 6
- 230000001568 sexual effect Effects 0.000 claims description 6
- 238000001962 electrophoresis Methods 0.000 claims description 5
- 238000010008 shearing Methods 0.000 claims description 5
- 238000010356 CRISPR-Cas9 genome editing Methods 0.000 claims description 4
- 238000001514 detection method Methods 0.000 claims description 4
- 239000013612 plasmid Substances 0.000 claims description 4
- 238000013518 transcription Methods 0.000 claims description 4
- 230000035897 transcription Effects 0.000 claims description 4
- 229920000936 Agarose Polymers 0.000 claims description 3
- 238000011746 C57BL/6J (JAX™ mouse strain) Methods 0.000 claims description 3
- 241000588724 Escherichia coli Species 0.000 claims description 3
- 101150063416 add gene Proteins 0.000 claims description 3
- 230000000694 effects Effects 0.000 claims description 3
- 230000002255 enzymatic effect Effects 0.000 claims description 3
- 238000001976 enzyme digestion Methods 0.000 claims description 3
- 238000000520 microinjection Methods 0.000 claims description 3
- 238000012795 verification Methods 0.000 claims description 3
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 3
- 239000003153 chemical reaction reagent Substances 0.000 claims description 2
- 108091028043 Nucleic acid sequence Proteins 0.000 claims 1
- 238000009395 breeding Methods 0.000 abstract description 2
- 230000001488 breeding effect Effects 0.000 abstract description 2
- 108090000623 proteins and genes Proteins 0.000 description 14
- 238000012217 deletion Methods 0.000 description 9
- 230000037430 deletion Effects 0.000 description 9
- 102000004169 proteins and genes Human genes 0.000 description 7
- 102000017278 Glutaredoxin Human genes 0.000 description 6
- 108050005205 Glutaredoxin Proteins 0.000 description 6
- 108091026890 Coding region Proteins 0.000 description 5
- RWSXRVCMGQZWBV-WDSKDSINSA-N glutathione Chemical compound OC(=O)[C@@H](N)CCC(=O)N[C@@H](CS)C(=O)NCC(O)=O RWSXRVCMGQZWBV-WDSKDSINSA-N 0.000 description 4
- 230000036542 oxidative stress Effects 0.000 description 4
- 102000011022 Chorionic Gonadotropin Human genes 0.000 description 3
- 108010062540 Chorionic Gonadotropin Proteins 0.000 description 3
- 108020004414 DNA Proteins 0.000 description 3
- 210000004027 cell Anatomy 0.000 description 3
- 230000006378 damage Effects 0.000 description 3
- 230000036541 health Effects 0.000 description 3
- 238000002347 injection Methods 0.000 description 3
- 239000007924 injection Substances 0.000 description 3
- 235000016709 nutrition Nutrition 0.000 description 3
- 230000035764 nutrition Effects 0.000 description 3
- 230000001105 regulatory effect Effects 0.000 description 3
- 238000011160 research Methods 0.000 description 3
- 108060008226 thioredoxin Proteins 0.000 description 3
- 241000894006 Bacteria Species 0.000 description 2
- CURLTUGMZLYLDI-UHFFFAOYSA-N Carbon dioxide Chemical compound O=C=O CURLTUGMZLYLDI-UHFFFAOYSA-N 0.000 description 2
- 108020005004 Guide RNA Proteins 0.000 description 2
- 108010050904 Interferons Proteins 0.000 description 2
- 102000014150 Interferons Human genes 0.000 description 2
- 102100036407 Thioredoxin Human genes 0.000 description 2
- 210000000805 cytoplasm Anatomy 0.000 description 2
- 201000010099 disease Diseases 0.000 description 2
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 2
- 210000002257 embryonic structure Anatomy 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 229960003180 glutathione Drugs 0.000 description 2
- 229940084986 human chorionic gonadotropin Drugs 0.000 description 2
- 238000003780 insertion Methods 0.000 description 2
- 230000037431 insertion Effects 0.000 description 2
- 229940079322 interferon Drugs 0.000 description 2
- 230000001404 mediated effect Effects 0.000 description 2
- 230000035772 mutation Effects 0.000 description 2
- 230000009437 off-target effect Effects 0.000 description 2
- 239000000829 suppository Substances 0.000 description 2
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 1
- 208000030507 AIDS Diseases 0.000 description 1
- 208000035143 Bacterial infection Diseases 0.000 description 1
- 238000010453 CRISPR/Cas method Methods 0.000 description 1
- 108090000695 Cytokines Proteins 0.000 description 1
- 102000004127 Cytokines Human genes 0.000 description 1
- 102000053602 DNA Human genes 0.000 description 1
- BWGNESOTFCXPMA-UHFFFAOYSA-N Dihydrogen disulfide Chemical compound SS BWGNESOTFCXPMA-UHFFFAOYSA-N 0.000 description 1
- 208000005189 Embolism Diseases 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 241000283073 Equus caballus Species 0.000 description 1
- 108010024636 Glutathione Proteins 0.000 description 1
- 108010003272 Hyaluronate lyase Proteins 0.000 description 1
- 102000001974 Hyaluronidases Human genes 0.000 description 1
- 108020005198 Long Noncoding RNA Proteins 0.000 description 1
- 241000124008 Mammalia Species 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 108700011259 MicroRNAs Proteins 0.000 description 1
- 108091005461 Nucleic proteins Proteins 0.000 description 1
- 102000004316 Oxidoreductases Human genes 0.000 description 1
- 108090000854 Oxidoreductases Proteins 0.000 description 1
- 238000011529 RT qPCR Methods 0.000 description 1
- 108020004682 Single-Stranded DNA Proteins 0.000 description 1
- 238000010459 TALEN Methods 0.000 description 1
- 102000002933 Thioredoxin Human genes 0.000 description 1
- 108010043645 Transcription Activator-Like Effector Nucleases Proteins 0.000 description 1
- 108010073062 Transcription Activator-Like Effectors Proteins 0.000 description 1
- 241000700605 Viruses Species 0.000 description 1
- 208000027418 Wounds and injury Diseases 0.000 description 1
- 210000000683 abdominal cavity Anatomy 0.000 description 1
- 125000000539 amino acid group Chemical group 0.000 description 1
- 230000006907 apoptotic process Effects 0.000 description 1
- 208000022362 bacterial infectious disease Diseases 0.000 description 1
- 230000004071 biological effect Effects 0.000 description 1
- 230000008827 biological function Effects 0.000 description 1
- 229910002092 carbon dioxide Inorganic materials 0.000 description 1
- 239000001569 carbon dioxide Substances 0.000 description 1
- 101150038500 cas9 gene Proteins 0.000 description 1
- 230000010261 cell growth Effects 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 229940015047 chorionic gonadotropin Drugs 0.000 description 1
- 210000000349 chromosome Anatomy 0.000 description 1
- 238000003776 cleavage reaction Methods 0.000 description 1
- 238000005520 cutting process Methods 0.000 description 1
- 230000001086 cytosolic effect Effects 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 235000005911 diet Nutrition 0.000 description 1
- 230000000378 dietary effect Effects 0.000 description 1
- 230000005782 double-strand break Effects 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 239000012634 fragment Substances 0.000 description 1
- 230000006870 function Effects 0.000 description 1
- 238000012239 gene modification Methods 0.000 description 1
- 238000010362 genome editing Methods 0.000 description 1
- 230000037308 hair color Effects 0.000 description 1
- 230000013632 homeostatic process Effects 0.000 description 1
- 229960002773 hyaluronidase Drugs 0.000 description 1
- 230000036039 immunity Effects 0.000 description 1
- 230000002401 inhibitory effect Effects 0.000 description 1
- 208000014674 injury Diseases 0.000 description 1
- 238000011813 knockout mouse model Methods 0.000 description 1
- 210000004962 mammalian cell Anatomy 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 239000002679 microRNA Substances 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000010172 mouse model Methods 0.000 description 1
- 108020004707 nucleic acids Proteins 0.000 description 1
- 102000039446 nucleic acids Human genes 0.000 description 1
- 150000007523 nucleic acids Chemical class 0.000 description 1
- 210000004940 nucleus Anatomy 0.000 description 1
- 210000003101 oviduct Anatomy 0.000 description 1
- 230000003647 oxidation Effects 0.000 description 1
- 238000007254 oxidation reaction Methods 0.000 description 1
- 230000004983 pleiotropic effect Effects 0.000 description 1
- 229920001184 polypeptide Polymers 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 102000004196 processed proteins & peptides Human genes 0.000 description 1
- 108090000765 processed proteins & peptides Proteins 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 238000006479 redox reaction Methods 0.000 description 1
- 230000008439 repair process Effects 0.000 description 1
- 230000007017 scission Effects 0.000 description 1
- 230000019491 signal transduction Effects 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 230000004083 survival effect Effects 0.000 description 1
- 125000003396 thiol group Chemical group [H]S* 0.000 description 1
- 229940094937 thioredoxin Drugs 0.000 description 1
- 238000002054 transplantation 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
- C12N15/87—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
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/0004—Oxidoreductases (1.)
- C12N9/0051—Oxidoreductases (1.) acting on a sulfur group of donors (1.8)
-
- 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
-
- 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
- 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
- C12N15/1137—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 enzymes
-
- 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
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
- C12N9/22—Ribonucleases RNAses, DNAses
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y108/00—Oxidoreductases acting on sulfur groups as donors (1.8)
- C12Y108/01—Oxidoreductases acting on sulfur groups as donors (1.8) with NAD+ or NADP+ as acceptor (1.8.1)
- C12Y108/01008—Protein-disulfide reductase (1.8.1.8), i.e. thioredoxin
-
- 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]
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Engineering & Computer Science (AREA)
- Chemical & Material Sciences (AREA)
- Zoology (AREA)
- Organic Chemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Wood Science & Technology (AREA)
- Molecular Biology (AREA)
- Biotechnology (AREA)
- General Engineering & Computer Science (AREA)
- Biomedical Technology (AREA)
- General Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Microbiology (AREA)
- Environmental Sciences (AREA)
- Medicinal Chemistry (AREA)
- Physics & Mathematics (AREA)
- Biophysics (AREA)
- Plant Pathology (AREA)
- Animal Behavior & Ethology (AREA)
- Veterinary Medicine (AREA)
- Animal Husbandry (AREA)
- Biodiversity & Conservation Biology (AREA)
- Mycology (AREA)
- Cell Biology (AREA)
- Virology (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
本发明公开了一种基于CRISPR/Cas9技术的Glrx1基因敲除动物模型的构建方法。包括以下步骤：一、靶向小鼠Glrx1基因的gRNA的选择和设计。二、sgRNA载体构建。三、sgRNA体外转录。四、小鼠一细胞期受精卵注射。五、F0代小鼠出生和鉴定。六、阳性F0代小鼠配繁，F1代小鼠出生与鉴定。通过本发明方法，能够成功获得Glrx1基因敲除动物模型。
Description
技术领域
本发明属于利用基因修饰技术制作基因敲除动物模型的领域，具体涉及一种基于CRISPR/Cas9技术的Glrx1基因敲除动物模型的构建方法。
背景技术
CRISPR/Cas(Clustered Regularly Interspaced Shot Palindromic repeats/CRISPR-associated)***，是一种来源于细菌获得性免疫的由RNA介导Cas蛋白对目的基因进行靶向修饰的技术。经过研究者改造过的Type II CRISPR/Cas9***自2013年成功敲除哺乳动物细胞后，现在己经被应用于多种模式生物的基因敲除。CRISPR/Cas9***载体构建简单快速、易操作、省时省力周期短，且几乎对所有物种都适用。CRISPR/Cas9和TALEN(Transcription Activator-like Effector Nucleases)的作用都是实现染色体上特定位点的双链断裂，然后引发自主损伤修复，修复会引发***或缺失，从而造成基因序列永久的缺失，即基因敲除。针对每个基因，CRISPR/Cas9只需要构建一个sgRNA(single guideRNA)，而且效率都很高，序列选择限制较小，只需要基因组上出现GG就可以。与zinc-fingernucleases(ZFNs)and TALEN相比，CRISPR/Cas9***具有相同或者更高的基因编辑效率，更便宜。相对于TALEN，CRISPR/Cas9引起的脱靶效应较高，但是使用成对sgRNA/Cas9-D10A>截短的sgRNA或者FoKI-dCas9可以极大的降低脱靶效应。目前，CRISPR/Cas9主要应用于基因定点突变(***或缺失)、基因定点敲入、两位点同时突变、小片段的缺失、编码基因和非编码基因(lncRNA、microRNA)的靶向基因敲除。
谷氧还蛋白(glutaredoxin,Glrx)普遍存在于细菌、病毒和哺乳动物体内，其表达受干扰素(interferon，IFN)调控，分子量为12kDa，由106-107个氨基酸残基组成，是硫氧还蛋白(thioredoxin,Trx)家族的重要分支，作为电子供体，参与组成巯基-二硫键氧化还原酶家族，依靠谷胱甘肽(GSH)将氧化状态的蛋白质二硫键还原为巯基，以维持细胞氧化还原稳态，在细胞信号转导过程中发挥重要作用。有文献报道，在氧化应激引起的损伤中，蛋白质氧化损伤先于核酸，蛋白发生羰基化和糖基化，从而失去生物活性。大量研究表明Glrx1是一种具有多种生物学功能的多效性细胞因子，与调节氧化还原反应、细胞生长和抑制凋亡有密切关系，与人类某些疾病，如获得性免疫缺陷综合征和细菌感染等的发生、发展也相关。
谷氧还蛋白是机体内能特异、高效的还原谷胱甘肽化蛋白质的一种酶蛋白，Glrx特异的恢复氧化应激损伤产生的谷胱甘肽化蛋白活性的能力可能会使其成为热点药物。构建Glrx1基因敲除小鼠模型，对于研究氧化应激、营养健康等具有重要意义。但传统的基因敲除方法成功率极低，一直未得到应用。近年来，CRISPR/Cas9技术得到广泛应用，为Glrx1基因敲除模型鼠的构建及其在营养与健康研究中的应用提供了可能性。
发明内容
本发明的目的是提供一种基于CRISPR/Cas9技术的Glrx1基因敲除动物模型的构建方法。
本发明的目的通过以下技术方案实现。
一种基于CRISPR/Cas9技术的Glrx1基因敲除动物模型的构建方法，包含以下步骤：
步骤一：靶向小鼠Glrx1基因的gRNA的选择和设计
在Glrx1内含子相应位置设计相应的sgRNA，其引物序列如SEQ ID NO.1和SEQ IDNO.2所示；
步骤二：sgRNA载体构建
首先BsaI酶切pUC57-sgRNA载体，37℃水浴1h后，1％的琼脂糖电泳，回收酶切产物；然后将sgRNA引物进行退火；最后，连接退火产物与回收的酶切产物，转化大肠杆菌，挑选单克隆进行PCR、PCR结果呈阳性送测序验证，得到正确的sgRNA载体；
步骤三：采用转录试剂盒，体外转录sgRNA和Cas9 mRNA，转录好的sgRNA备用；试剂盒名称：AM1354+AM1908,Ambion by Life Technologies；
步骤四：Cas9 sgRNA体系(Cas9 mRNA和sgRNA)的受精卵显微注射；Cas9表达质粒为cas9 D10A(plasmid#42335)，Addgene；
步骤五：F0代小鼠出生与鉴定；
步骤六：F0小鼠性成熟配繁，F1代小鼠鉴定。
其中个，步骤六优选：F0代小鼠在性成熟后和C57BL/6J小鼠回交进行配繁，出生F1代小鼠在1周龄进行剪尾鉴定，得到阳性的F1代杂合子。
进一步优选从mRNA水平和酶解测序来鉴定F1代。
一种基于CRISPR-Cas9基因敲除技术的Glrx1基因敲除试剂盒，包括：
1)sgRNA载体，所述的sgRNA载体以pUC57-sgRNA载体为出发载体，含针对Glrx1基因的sgRNA；该sgRNA由SEQ ID NO.1和SEQ ID NO.2所示的sgRNA引物退火得到；
2)以及配套的检测试剂，用于检测Glrx1基因的剪切效果和评估基因敲除效率。
本发明所述的基于CRISPR-Cas9基因敲除技术的Glrx1基因敲除试剂盒，优选还包含Cas9 mRNA或用于表达Cas9 mRNA的Cas9表达质粒。
有益效果：
本实验难点其一在于sgRNA序列定位，本实验采用的sgRNA序列高效，不易脱靶；其二在于对Cas9 sgRNA体系的优化，使小鼠后代阳性率更高，脱靶率低。应用该技术制作的Glrx1敲除小鼠解决了传统基因敲除技术中基因脱靶率高、动物成活率低等瓶颈问题，可广泛应用在膳食营养与健康、氧化应激及相关疾病的研究中的应用。
附图说明
图1、Glrx-1-Cas9-KO小鼠策略设计图
图2、sgRNA载体图谱
图3、PCR检测策略
图4、61#，62#，64#电泳结果
图5、73#，74#，75#电泳结果
图6、7周龄雄性纯合Glrx1-/-小鼠照片
具体实施方式
实施例1
基于CRISPR/Cas9技术的Glrx1基因敲除动物模型的构建方法通过以下步骤实现：
步骤一：靶向小鼠Glrx1基因的gRNA的选择和设计
设计Glrx-1-Cas9-KO小鼠策略，如图1所示。根据策略，设计相应sgRNA序列，根据策略，在Glrx-1内含子相应位置设计相应的sgRNA，订购相应Oligo；sgRNA序列如下：
| sgRNA名称 | 序列 | PAM |
| Glrx-3S1(forward) | CGGAGATGACACTTACTGATGGG(SEQ ID NO.1) | GGG |
| Glrx-5S1(forward) | GCTAAGCGCCGCTGCATTACCGG(SEQ ID NO.2) | CGG |
步骤二：sgRNA载体构建
首先BsaI酶切pUC57-sgRNA载体，37℃水浴1h后，1％的琼脂糖电泳，回收酶切产物。然后将订购的sgRNA引物进行退火。最后，连接退火产物与回收的酶切产物，转化大肠杆菌，挑选单克隆进行PCR，PCR结果呈阳性送测序验证，得到正确的sgRNA载体，载体图谱如图2所示。
步骤三：sgRNA体外转录
采用转录试剂盒，体外转录sgRNA和Cas9 mRNA，转录好的sgRNA和Cas9 mRNA备用。试剂盒名称：AM1354+AM1908,购自Ambion公司.
步骤四：受精卵显微注射
1.准备单细胞受精卵
小鼠超排：第一天，腹腔注射马绒毛膜***5IU/只，46-48小时后注射人绒毛膜***，注射完人绒毛膜***后将2只雌鼠与单放雄鼠合笼。第四天上午检栓，见栓的记为0.5天。
获取受精卵：脱颈椎处死见栓0.5天的小鼠，剪出输卵管，用显微镊取出成团的卵子，透明质酸酶消化后，挑选形态饱满、胞质均匀的胚胎于M16中培养。
2.显微注射受精卵
将挑选的受精卵转移入准备好的M2条带中，排成一列(30-50枚左右)。将注射皿放在倒置显微镜的载物台上，使M2液滴长条的方向与操作者垂直，即位于y轴上。将注射管刺入胞浆内，注入Cas9 sgRNA体系(sgRNA和Cas9 mRNA)，Cas9表达质粒为cas9 D10A(plasmid#42335)，Addgene；见到胞质松散后迅速退针。注射结束后,将胚胎转移至含有M16培养液的培养皿中，放入37℃、5％二氧化碳培养箱恢复0.5-1.0小时。将受精卵移植到E0.5天假孕受体内。移植后大约19-21天出生F0代小鼠。
步骤五：F0代小鼠出生与鉴定
出生小崽数量为39只，存活数量38只，F0代小鼠出生1周后进行剪尾鉴定，得到7只阳性F0代小鼠，毛色为黑色，性别为5雌2雄，图6为F1代两个雄性纯合Glrx1-/-小鼠照片。
PCR反应体系：
PCR检测策略如图3所示。
步骤六：F0小鼠性成熟配繁，F1代小鼠鉴定
F0代小鼠在8周龄左右性成熟和C57BL/6J小鼠回交进行配繁，出生F1代小鼠在1周龄进行剪尾鉴定，得到6只阳性的F1代杂合子，列表如下：
| 序号 | 性别 | 颜色 | 基因型 | 雌/雄 | 代数 |
| 61 | ♂ | 黑 | -7588bp/wt,E1-E2(整个编码区)全部删除 | ♂14 | F1 |
| 62 | ♂ | 黑 | -7588bp/wt,E1-E2(整个编码区)全部删除 | ♂14 | F1 |
| 64 | ♀ | 黑 | -7588bp/wt,E1-E2(整个编码区)全部删除 | ♂14 | F1 |
| 73 | ♀ | 黑 | -7898bp/wt,E1-E2(整个编码区)全部删除 | ♀7 | F1 |
| 74 | ♀ | 黑 | -7898bp/wt,E1-E2(整个编码区)全部删除 | ♀7 | F1 |
| 75 | ♀ | 黑 | -7898bp/wt,E1-E2(整个编码区)全部删除 | ♀7 | F1 |
分别从mRNA水平和酶解测序来鉴定F1代，mRNA水平所采用qPCR手段来实现，条件同上。从测序结果可看出61#,62#,64#,73#,74#,75#与野生型对比序列长度至少少了-7588bp，也就是说明E1-E2被删掉。
61#，62#，64#：
GCCCTTTAAAACTGAAGCATCCTACTTGGTAACTCCTCCTCCAAGGAGGTTCCTTATTAAATGAGAGCTGCTGGCTAAGCGCC--------------7588bp----------ATACACATAGTTCTAGACATAAATACACAAAAAGATAACGT
73#，74#，75#：
CCAGTGTGCAATGGTAGGCCTAGGAAGTACTGACTCATACCAA--------------7898bp---------TAGCTAAGGATGGAAATTTGGGAAGTAT
实施例2
本实施例与实施例1的不同点在于步骤三中所用单链DNA模板和引物序列为2074-Glrx-gtF1。其它步骤与实施例1相同；结果均与实施例1相同。
实施例3
本实施例与实施例1的不同点在于步骤四中合笼雄鼠的品种优选为C57BL/6J雄鼠。其它步骤与实施例1相同。
<110> 南京农业大学
<120> 一种基于CRISPR/Cas9技术的Glrx1基因敲除动物模型的构建方法
<160> 2
<210> 1
<211> 23
<212> DNA
<213> 人工序列
<220>
<223> 引物Glrx-3S1
<400> 1
cggagatgac acttactgat ggg 23
<210> 2
<211> 23
<212> DNA
<213> 人工序列
<220>
<223> 引物Glrx-5S1
<400> 2
gctaagcgcc gctgcattac cgg 23
Claims (5)
1.一种基于CRISPR/Cas9技术的Glrx1基因敲除动物模型的构建方法，其特征在于包含以下步骤：
步骤一：靶向小鼠Glrx1基因的sgRNA的选择和设计
在Glrx1内含子相应位置设计相应的sgRNA，所述sgRNA引物的核苷酸序列如SEQ IDNO.1和SEQ ID NO.2所示；
步骤二：sgRNA载体构建
首先BsaI酶切pUC57-sgRNA载体，37℃水浴1h后，1%的琼脂糖电泳，回收酶切产物；然后将步骤一中所述的sgRNA引物进行退火；最后，连接退火产物与回收的酶切产物，转化大肠杆菌，挑选单克隆进行PCR、PCR结果呈阳性送测序验证，得到正确的sgRNA载体；
步骤三：采用转录试剂盒，体外转录sgRNA和Cas9 mRNA，转录好的sgRNA备用；其中，Cas9表达质粒为Addgene plasmid # 42335；
步骤四：Cas9 mRNA和sgRNA 组成的Cas9 sgRNA体系的小鼠受精卵显微注射，其中，Cas9表达质粒为Addgene plasmid # 42335；
步骤五：F0代小鼠出生与鉴定；
步骤六：F0小鼠性成熟配繁，F1代小鼠鉴定。
2.根据权利要求1所述的构建方法，其特征在于步骤六：F0代小鼠在性成熟后和C57BL/6J小鼠回交进行配繁，出生F1代小鼠在1周龄进行剪尾鉴定，得到阳性的F1代杂合子。
3.根据权利要求2所述的构建方法，其特征在于从mRNA水平和酶解测序来鉴定F1代小鼠。
4.一种基于 CRISPR-Cas9 基因敲除技术的Glrx1基因敲除试剂盒，其特征在于包括 ：
1) sgRNA载体，所述的sgRNA载体以pUC57-sgRNA载体为出发载体，含针对Glrx1基因的sgRNA；该sgRNA由SEQ ID NO.1和SEQ ID NO.2所示的sgRNA引物退火得到；
2)以及配套的检测试剂，用于检测 Glrx1基因的剪切效果和评估基因敲除效率。
5.根据权利要求4所述的基于 CRISPR-Cas9 基因敲除技术的Glrx1基因敲除试剂盒，其特征在于还包含Cas9 mRNA或用于表达Cas9 mRNA的Cas9表达质粒。
Priority Applications (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710390894.3A CN107287245B (zh) | 2017-05-27 | 2017-05-27 | 一种基于CRISPR/Cas9技术的Glrx1基因敲除动物模型的构建方法 |
| GB1918320.1A GB2578026B (en) | 2017-05-27 | 2018-05-03 | Method for constructing GLRX1 gene knock-out animal model based on CRISPR/CAS9 |
| PCT/CN2018/085432 WO2018219093A1 (zh) | 2017-05-27 | 2018-05-03 | 一种基于CRISPR/Cas9技术的Glrx1基因敲除动物模型的构建方法 |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710390894.3A CN107287245B (zh) | 2017-05-27 | 2017-05-27 | 一种基于CRISPR/Cas9技术的Glrx1基因敲除动物模型的构建方法 |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN107287245A CN107287245A (zh) | 2017-10-24 |
| CN107287245B true CN107287245B (zh) | 2020-03-17 |
Family
ID=60095140
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201710390894.3A Active CN107287245B (zh) | 2017-05-27 | 2017-05-27 | 一种基于CRISPR/Cas9技术的Glrx1基因敲除动物模型的构建方法 |
Country Status (3)
| Country | Link |
|---|---|
| CN (1) | CN107287245B (zh) |
| GB (1) | GB2578026B (zh) |
| WO (1) | WO2018219093A1 (zh) |
Families Citing this family (50)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP3613852A3 (en) | 2011-07-22 | 2020-04-22 | President and Fellows of Harvard College | Evaluation and improvement of nuclease cleavage specificity |
| US20150044192A1 (en) | 2013-08-09 | 2015-02-12 | President And Fellows Of Harvard College | Methods for identifying a target site of a cas9 nuclease |
| US9359599B2 (en) | 2013-08-22 | 2016-06-07 | President And Fellows Of Harvard College | Engineered transcription activator-like effector (TALE) domains and uses thereof |
| US9737604B2 (en) | 2013-09-06 | 2017-08-22 | President And Fellows Of Harvard College | Use of cationic lipids to deliver CAS9 |
| US9322037B2 (en) | 2013-09-06 | 2016-04-26 | President And Fellows Of Harvard College | Cas9-FokI fusion proteins and uses thereof |
| US9228207B2 (en) | 2013-09-06 | 2016-01-05 | President And Fellows Of Harvard College | Switchable gRNAs comprising aptamers |
| US9068179B1 (en) | 2013-12-12 | 2015-06-30 | President And Fellows Of Harvard College | Methods for correcting presenilin point mutations |
| AU2015298571B2 (en) | 2014-07-30 | 2020-09-03 | President And Fellows Of Harvard College | Cas9 proteins including ligand-dependent inteins |
| CN108513575A (zh) | 2015-10-23 | 2018-09-07 | 哈佛大学的校长及成员们 | 核碱基编辑器及其用途 |
| WO2018027078A1 (en) | 2016-08-03 | 2018-02-08 | President And Fellows Of Harard College | Adenosine nucleobase editors and uses thereof |
| CA3033327A1 (en) | 2016-08-09 | 2018-02-15 | President And Fellows Of Harvard College | Programmable cas9-recombinase fusion proteins and uses thereof |
| WO2018039438A1 (en) | 2016-08-24 | 2018-03-01 | President And Fellows Of Harvard College | Incorporation of unnatural amino acids into proteins using base editing |
| KR20240007715A (ko) | 2016-10-14 | 2024-01-16 | 프레지던트 앤드 펠로우즈 오브 하바드 칼리지 | 핵염기 에디터의 aav 전달 |
| US10745677B2 (en) | 2016-12-23 | 2020-08-18 | President And Fellows Of Harvard College | Editing of CCR5 receptor gene to protect against HIV infection |
| EP3592853A1 (en) | 2017-03-09 | 2020-01-15 | President and Fellows of Harvard College | Suppression of pain by gene editing |
| JP2020510439A (ja) | 2017-03-10 | 2020-04-09 | プレジデント アンド フェローズ オブ ハーバード カレッジ | シトシンからグアニンへの塩基編集因子 |
| SG11201908658TA (en) | 2017-03-23 | 2019-10-30 | Harvard College | Nucleobase editors comprising nucleic acid programmable dna binding proteins |
| US11560566B2 (en) | 2017-05-12 | 2023-01-24 | President And Fellows Of Harvard College | Aptazyme-embedded guide RNAs for use with CRISPR-Cas9 in genome editing and transcriptional activation |
| CN107287245B (zh) * | 2017-05-27 | 2020-03-17 | 南京农业大学 | 一种基于CRISPR/Cas9技术的Glrx1基因敲除动物模型的构建方法 |
| US11732274B2 (en) | 2017-07-28 | 2023-08-22 | President And Fellows Of Harvard College | Methods and compositions for evolving base editors using phage-assisted continuous evolution (PACE) |
| US11319532B2 (en) | 2017-08-30 | 2022-05-03 | President And Fellows Of Harvard College | High efficiency base editors comprising Gam |
| CN111757937A (zh) | 2017-10-16 | 2020-10-09 | 布罗德研究所股份有限公司 | 腺苷碱基编辑器的用途 |
| CN108410906A (zh) * | 2018-03-05 | 2018-08-17 | 淮海工学院 | 一种适用于海洋甲壳类线粒体基因组的CRISPR/Cpf1基因编辑方法 |
| CN108642090A (zh) * | 2018-05-18 | 2018-10-12 | 中国人民解放军总医院 | 基于CRISPR/Cas9技术获得Nogo-B敲除模式小鼠的方法及应用 |
| CN109022485B (zh) * | 2018-08-16 | 2021-02-02 | 华东师范大学 | 一种视神经萎缩动物模型的构建方法、试剂盒和应用 |
| CN109777837A (zh) * | 2018-12-26 | 2019-05-21 | 首都医科大学 | 一种利用CRISPR/Cas9***构建致死基因全身性敲除小鼠模型的方法 |
| CN109694885B (zh) * | 2019-02-18 | 2021-03-02 | 中国人民解放军总医院 | 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 |
| DE112020001342T5 (de) | 2019-03-19 | 2022-01-13 | President and Fellows of Harvard College | Verfahren und Zusammensetzungen zum Editing von Nukleotidsequenzen |
| CN110157704B (zh) * | 2019-04-04 | 2021-08-06 | 中山大学 | 一种抗小鼠肝炎病毒的小鼠及其制备方法 |
| CN110218743B (zh) * | 2019-04-25 | 2021-05-04 | 天津市环湖医院(天津市神经外科研究所、天津市脑系科中心医院) | 一种基于CRISPR/Cas9技术的牛磺酸转运体基因敲除大鼠模型的构建方法 |
| CN110157744A (zh) * | 2019-06-13 | 2019-08-23 | 福建农林大学 | 一种蜜蜂CRISPR-Cas9基因编辑方法 |
| WO2021030271A2 (en) | 2019-08-09 | 2021-02-18 | Nutcracker Therapeutics, Inc. | Methods and apparatuses for manufacturing for removing material from a therapeutic composition |
| CN111109199B (zh) * | 2019-11-22 | 2022-02-18 | 温州医科大学附属第二医院、温州医科大学附属育英儿童医院 | Slc12a9基因敲除小鼠模型及其建立方法和用途 |
| CN111321172A (zh) * | 2019-12-27 | 2020-06-23 | 温州医科大学附属第二医院、温州医科大学附属育英儿童医院 | 一种利用CRISPR-Cas9技术建立CDK13基因敲除动物模型的方法 |
| CN111518837A (zh) * | 2020-04-02 | 2020-08-11 | 广州欣意生物技术有限公司 | 一种敲除adrb3基因的小鼠及其应用 |
| EP4146804A1 (en) | 2020-05-08 | 2023-03-15 | The Broad Institute Inc. | Methods and compositions for simultaneous editing of both strands of a target double-stranded nucleotide sequence |
| CN112662669A (zh) * | 2020-12-23 | 2021-04-16 | 成都药康生物科技有限公司 | 一种Il21基因敲除小鼠模型及其构建方法、应用 |
| CN112852803B (zh) * | 2021-02-05 | 2023-10-31 | 上海市第六人民医院 | 一种全身性eepd1敲除的动物模型构建方法及其应用 |
| CN112899279B (zh) * | 2021-03-08 | 2023-02-14 | 浙江大学 | 一种构建Fzd6基因敲除小鼠模型的方法及应用 |
| CN112980880B (zh) * | 2021-03-08 | 2023-05-02 | 浙江大学 | 基于CRISPR/Cas9构建Fzd6-Q152E定点突变小鼠模型的方法及应用 |
| CN113403311A (zh) * | 2021-06-18 | 2021-09-17 | 南方科技大学 | 一种靶向casc5基因的sgRNA及其应用 |
| CN113862266A (zh) * | 2021-09-18 | 2021-12-31 | 赛业（苏州）生物科技有限公司 | 靶向小鼠BBS５基因的gRNA及构建Bardet–Biedl小鼠模型的方法 |
| CN113755498A (zh) * | 2021-09-27 | 2021-12-07 | 赛业（苏州）生物科技有限公司 | 靶向小鼠Ube3a基因的gRNA及构建AS疾病小鼠模型的方法 |
| CN113957096A (zh) * | 2021-10-26 | 2022-01-21 | 上海交通大学医学院附属第九人民医院 | 一种Nf1基因敲除动物模型的构建方法 |
| CN114317603B (zh) * | 2022-01-12 | 2023-10-13 | 北京航空航天大学 | 一种Foxi3基因定点突变小鼠模型的构建方法及其应用 |
| CN115044619B (zh) * | 2022-05-25 | 2023-09-15 | 郑州大学第一附属医院 | 一种全身诱导性Ppp3ca基因敲除鼠模型的构建方法 |
| CN115058456B (zh) * | 2022-06-23 | 2023-09-19 | 五邑大学 | Hprt基因敲除的动物模型的构建方法和应用 |
| CN114958923A (zh) * | 2022-06-28 | 2022-08-30 | 赛业（苏州）生物科技有限公司 | 一种Nr1d1基因敲除小鼠动物模型的构建方法及其应用 |
| CN115992178A (zh) * | 2022-08-08 | 2023-04-21 | 首都医科大学附属北京天坛医院 | 核心蛋白聚糖转基因小鼠的制备方法及应用 |
| CN116656747B (zh) * | 2023-01-10 | 2024-01-26 | 广东医科大学附属医院 | 一种体内动态观察溶酶体损伤的方法 |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106282231A (zh) * | 2016-09-06 | 2017-01-04 | 陕西慧康生物科技有限责任公司 | 粘多糖贮积症ii型动物模型的构建方法及应用 |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107287245B (zh) * | 2017-05-27 | 2020-03-17 | 南京农业大学 | 一种基于CRISPR/Cas9技术的Glrx1基因敲除动物模型的构建方法 |
-
2017
- 2017-05-27 CN CN201710390894.3A patent/CN107287245B/zh active Active
-
2018
- 2018-05-03 GB GB1918320.1A patent/GB2578026B/en active Active
- 2018-05-03 WO PCT/CN2018/085432 patent/WO2018219093A1/zh active Application Filing
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106282231A (zh) * | 2016-09-06 | 2017-01-04 | 陕西慧康生物科技有限责任公司 | 粘多糖贮积症ii型动物模型的构建方法及应用 |
Non-Patent Citations (1)
| Title |
|---|
| Attenuated cardiovascular hypertrophy and oxidant generation in response to angiotensin II infusion in glutaredoxin-1 knockout mice;Markus M.Bachschmid等;《Free Radical Biology & Medicine》;20101015;第49卷(第7期);第1221-1229页，参见摘要 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN107287245A (zh) | 2017-10-24 |
| GB2578026A8 (en) | 2020-07-22 |
| GB201918320D0 (en) | 2020-01-29 |
| GB2578026A (en) | 2020-04-15 |
| GB2578026B (en) | 2022-11-23 |
| WO2018219093A1 (zh) | 2018-12-06 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN107287245B (zh) | 一种基于CRISPR/Cas9技术的Glrx1基因敲除动物模型的构建方法 | |
| CN106047930B (zh) | 一种PS1基因条件性敲除flox大鼠的制备方法 | |
| US20220015342A1 (en) | Gene editing of reproductive hormones to reduce fertility in ictalurus punctatus | |
| CN105861554B (zh) | 一种基于对Rbmy基因进行编辑来实现动物性别控制的方法和应用 | |
| CN110551759B (zh) | 一种提高转基因细胞重组效率的组合物及方法 | |
| CN105132427A (zh) | 一种以RNA介导的特异性敲除双基因获得基因编辑绵羊的方法及其专用sgRNA | |
| CN110643636B (zh) | 一种团头鲂MSTNa&b基因敲除方法与应用 | |
| WO2018030208A1 (ja) | 遺伝子ノックイン細胞の作製方法 | |
| CN113088521A (zh) | 一种基于CRISPR/Cas9技术的Ahnak2基因敲除动物模型的构建方法 | |
| CN105505879B (zh) | 一种培养转基因动物胚胎细胞或转基因动物的方法及培养基 | |
| CN113234756A (zh) | 一种基于CRISPR/Cas9技术的LAMA3基因敲除动物模型的构建方法 | |
| WO2011091562A1 (zh) | 一种培育猪生长激素表达量增强的转基因动物的方法 | |
| CN114250247B (zh) | Glud1突变基因敲入小鼠动物模型的构建方法及其应用 | |
| CN115261360A (zh) | 一种gata6基因敲除斑马鱼模型的构建方法 | |
| CN114958852A (zh) | 一种Ifnar1基因敲除小鼠动物模型的构建方法及其应用 | |
| CN111549070B (zh) | 对x染色体多拷贝基因进行编辑实现动物性别控制的方法 | |
| CN105132426A (zh) | 一种以RNA介导的特异性敲除FGF5基因获得基因编辑绵羊的方法及其专用sgRNA | |
| CN111849977B (zh) | 一种***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 | |
| CN113462692B (zh) | 一种适于翘嘴鲌受精卵Mstn基因敲除的方法及应用 | |
| CN111518839B (zh) | 一种等位特异性位点编辑方法 | |
| CN110643605B (zh) | 一种团头鲂MSTNa&b基因敲除的gRNA及其模板 | |
| CN111500580B (zh) | 一种基因编辑方法 | |
| CN117821467A (zh) | 一种Mfsd2a基因特异性敲入小鼠肝组织模型的构建方法 | |
| CN115992178A (zh) | 核心蛋白聚糖转基因小鼠的制备方法及应用 | |
| CN116555345A (zh) | 用于研究gne肌病的动物模型的方法和应用 |
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 |