CN114958908A - 基于CRISPR/Cas9构建Ets2基因超级增强子敲除动物模型的方法及其应用 - Google Patents

基于CRISPR/Cas9构建Ets2基因超级增强子敲除动物模型的方法及其应用 Download PDF

Info

Publication number
CN114958908A
CN114958908A CN202111674779.1A CN202111674779A CN114958908A CN 114958908 A CN114958908 A CN 114958908A CN 202111674779 A CN202111674779 A CN 202111674779A CN 114958908 A CN114958908 A CN 114958908A
Authority
CN
China
Prior art keywords
mouse
sets2
animal model
knockout
cas9
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
Application number
CN202111674779.1A
Other languages
English (en)
Other versions
CN114958908B (zh
Inventor
秦利涛
张倩
郭正隆
郝冰涛
王红丹
张晓梅
王鑫
康冰
张安
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Henan Provincial Peoples Hospital
Original Assignee
Henan Provincial Peoples Hospital
Priority date (The priority date 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 date listed.)
Filing date
Publication date
Application filed by Henan Provincial Peoples Hospital filed Critical Henan Provincial Peoples Hospital
Priority to CN202111674779.1A priority Critical patent/CN114958908B/zh
Publication of CN114958908A publication Critical patent/CN114958908A/zh
Application granted granted Critical
Publication of CN114958908B publication Critical patent/CN114958908B/zh
Active legal-status Critical Current
Anticipated expiration legal-status Critical

Links

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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/82—Translation products from oncogenes
    • 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
    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C12N9/22—Ribonucleases RNAses, DNAses
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • 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
    • A01K2217/00—Genetically modified animals
    • A01K2217/15—Animals comprising multiple alterations of the genome, by transgenesis or homologous recombination, e.g. obtained by cross-breeding
    • 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
    • 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
    • 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
    • C12N2810/00—Vectors comprising a targeting moiety
    • C12N2810/10—Vectors comprising a non-peptidic targeting moiety

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Engineering & Computer Science (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Zoology (AREA)
  • Biomedical Technology (AREA)
  • Biotechnology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Wood Science & Technology (AREA)
  • General Engineering & Computer Science (AREA)
  • Molecular Biology (AREA)
  • General Health & Medical Sciences (AREA)
  • Biochemistry (AREA)
  • Biophysics (AREA)
  • Microbiology (AREA)
  • Plant Pathology (AREA)
  • Physics & Mathematics (AREA)
  • Environmental Sciences (AREA)
  • Veterinary Medicine (AREA)
  • Medicinal Chemistry (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Animal Behavior & Ethology (AREA)
  • Animal Husbandry (AREA)
  • Biodiversity & Conservation Biology (AREA)
  • Oncology (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

本发明属于生物工程和基因编辑技术领域，具体涉及一种基于CRISPR/Cas9构建Ets2基因超级增强子敲除动物模型的方法及其应用。SEts2敲除动物模型的构建方法包括如下步骤：1)靶向小鼠SEts2序列gRNA设计：2)将步骤1)中的一对gRNA与cas9质粒共同注射小鼠胚胎，获得F0代小鼠；3)鉴定SEts2基因型，筛选出阳性F0代小鼠；4)阳性F0代小鼠与野生型小鼠杂交得到F1代小鼠，F1代杂合小鼠自交获得纯合后代，从而构建得到SEts2基因敲除的动物模型。本发明基于CRISPR/Cas9技术实现对SEts2的定点敲除，具有操作简单、敲除效率高的优势。

Description

基于CRISPR/Cas9构建Ets2基因超级增强子敲除动物模型的 方法及其应用
技术领域
本发明属于生物工程、基因编辑和实验动物模型技术领域，具体涉及一种基于CRISPR/Cas9构建Ets2基因超级增强子敲除动物模型的方法及其应用。
背景技术
Ets2基因属于转绿因子(Transcription Factors,TFs)ETS家族成员，因为包含可以与DNA结合并且调节转录的ETS结构域而被命名。ETS家族是目前所知最大的信号依赖的转录因子家族之一，该家族包含30多个成员，其通过调节细胞的增殖与分化、细胞凋亡与衰老等作用，参与众多生理和病理过程。Ets2基因编码E26转录因子2(E26 oncogene homolog2)，可以调控众多的下游靶基因，从而调节效应细胞的增值、分化及凋亡。近期研究表明转录因子Ets2可以帮助建立和维持染色体的空间构象，影响基因转录，从而调控细胞的行为及功能。然而，Ets2基因受到哪些顺势调控元件的调控仍不清楚。
超级增强子(Super Enhancer，SE)是最近发现的一类基因组调控序列，可以调节细胞类型特异性基因表达，与细胞发育和分化密切相关，影响肿瘤、糖尿病、自身免疫疾病等人类常见疾病。超级增强子通常距离受调控基因线性距离比较远，研究发现它们在空间上与启动子靠近，从而调控靶基因的表达。研究发现ETS2基因附近存在超级增强子SuperEnhancer-Ets2(SEts2)，该增强子是否参与ETS2的调控，如何参与ETS2调控，这些仍不清楚。因此，通过CRISPR/Cas9基因编辑技术构建SEst2序列敲除小鼠，对研究ETS2基因表达至关重要。
基因组编辑技术(Genome editing technology)是通过人工手段在基因组水平时对DNA序列进行改造的遗传操作技术，包括特定DNA片段的插入、敲除、替換和点突变。其主要原理是在基因组的特定位置产生双链DNA断裂(Double-suranded break,DSB)后通过非同源末端连接(Non-homologous end joining,NHE)或同源重组(Homologousrecombination,HR)的方式进行修复。
随着核酸研究的深入，基因编辑技术先后经历了锌指核酸酶(zinc-fingernuclease，ZNF)、转录激活因子样效应物核酸酶(Transcription Activator-likeEffector Nucleases,TALEN)及成簇规律短回文重复序列相关蛋白(Clustered RegularlyInterspaced Shot Palindromic repeats associated proteins,CRISPR/Cas)。由于CRISPR/Cas技术与其之前的基因编辑技术相比具有更高的灵活性、省时、操作简单等优势，成为基因编辑技术的主流。本发明就是基于CRISPR/Cas9技术对C57/BL小鼠的Ets2基因超级增强子(SEts2)进行编辑，从而得到SEts2纯合敲除小鼠，为研究超级增强子SEts2对Ets2基因的调控功能提供合适的动物模型。
发明内容
本发明旨在提供一种基于CRISPR/Cas9构建Ets2基因超级增强子敲除动物模型的方法及其应用。
针对上述目的，本发明采用的技术方案如下：
第一方面，本发明提供基于CRISPR/Cas9构建Ets2基因超级增强子敲除动物模型的方法，包括如下步骤：
1)靶向C57小鼠SEts2序列的gRNA设计：
根据SEts2序列设计一对相应的gRNA，其中，gRNA的序列如SEQ ID NO.1和SEQ IDNO.2所示；
SEts2序列的核苷酸序列位于Chr16:95721049-95979933；
2)mRNA制备并显微注射获得F0小鼠：
将步骤1)中的一对gRNA在体外逆转录为mRNA，并将mRNA和cas9质粒共同注射小鼠胚胎，获得F0代小鼠；
3)鉴定SEst2基因，筛选出阳性F0代小鼠；
4)阳性F0代小鼠与野生型小鼠杂交得到F1代小鼠，F1代杂合小鼠自交获得纯合后代，从而构建得到SEst2基因敲除的动物模型。
进一步地，步骤3)中用于SEst2基因鉴定的引物包括公用上游引物和两条下游引物，其中，上游引物序列如SEQ ID NO.3所示，下游引物序列如SEQ ID NO.4和EQ ID NO.5所示。
第二方面，本发明提供一种由上述方法构建得到的SEst2敲除的动物模型。
第三方面，本发明构建的SEst2敲除的动物模型可应用于多种基础医学实验，包括但不限于，肿瘤、发育、遗传病等，另外也可以用来研究超级增强子对Ets2基因表达的调控。
与现有技术相比，本发明的有益效果如下：
本发明基于CRISPR/Cas9实现对SEst2的定点敲除，具有敲除效率高的优势，所构建的SEst2敲除的动物模型有助于研究多种疾病，并可以用来研究超级增强子对Ets2的表达调控。
附图说明
图1为小鼠SEst2结构示意图；
图2为用于SEst2基因型鉴定引物的设计方案图；
图3为F0代小鼠基因型鉴定琼脂糖电泳图；
图4为F0代小鼠测序鉴定结果图。
具体实施方式
为更好地说明本发明的目的、技术方案和优点，下面将结合附图和具体实施例对本发明作进一步说明。本领域技术人员应当理解，此处所描述的具体实施例仅用以解释本发明，并不用于限定本发明。
实施例中所用的试验方法如无特殊说明，均为常规方法；所用的材料、试剂等，如无特殊说明，均可从商业途径得到。
实施例1
一种基于CRISPR/Cas9技术构建SEst2敲除动物模型的方法，包括如下步骤：
1.靶向小鼠SEst2调控序列的gRNA设计
在SEst2区域两侧相应位置设计一对用于SEts2基因敲除的gRNA(gRNA1和gRNA2)，其中，gRNA1和gRNA2的位置如图1所示，其序列如表1所示。其中，靶向SEst2核苷酸序列位于小鼠Chr16:95721049-95979933之间，gRNA1与gRNA2分别位于该区间的上游与下游。
表1用于小鼠SEts2序列敲除的gRNA序列
gRNA名称 SEQ ID NO gRNASequence(5'to3')
gRNA1 SEQ ID NO.1 AAGACGACTTGGTCACACGC
gRNA2 SEQ ID NO.2 AGGACCTAGTGAGGACCAAT
2.mRNA制备并显微注射获得F0小鼠
将步骤1)中的gRNA1和gRNA2进行纯化，将纯化后的产物和cas9质粒(Cas9质粒由发明人所在实验室改造得到)共同注射C57小鼠胚胎，注射后将胚胎移植到代孕受体小鼠的输卵管内，原核注射共获得240枚胚胎。
3.F0代小鼠的出生及鉴定
出生小崽数量为16只，F0代小鼠出生1周后进行剪尾鉴定，得到2只阳性F0代小鼠，毛色为黑色，2只阳性F0代小鼠均为雄鼠，如表2所示。
表2阳性F0代小鼠信息列表
出生日期 数量 编号及性别
2018/6/24 2 ♂1474、1476
用于SEts2基因型鉴定的特异性引物设计方案如图2所示，野生型(WT)与SEts2基因敲除型(KO)采用共同的上游引物，野生型采用SEts2-seqF作为上游引物，SEts2-seqR1作为下游引物；SEts2基因敲除型采用SEts2-seqF作为上游引物，SEts2-seqR2作为下游引物。SEts2-seqF、SEts2-seqR1和SEts2-seqR2的序列分别如表3中的SEQ ID NO.3、SEQ IDNO.4、SEQ ID NO.5所示。
表3用于SEts2基因型鉴定的特异性引物
Figure BDA0003450726840000041
对F0代小鼠出生1周后进行剪尾鉴定的PCR反应体系和反应程序如表4和表5所示，PCR反应后野生型与SEts2基因敲除型中针对SEts2基因的PCR产物大小分别为737bp和935bp，其中对F0代小鼠进行基因型判断的标准如表6所示，由SEts2-seqF和SEts2-seqR1进行PCR扩增后获得737bp的产物，而由SEts2-seqF和SEts2-seqR2进行PCR扩增后未获得任何产物，则认定该小鼠为野生型小鼠；当由SEts2-seqF和SEts2-seqR1进行PCR扩增后未获得任何产物，而由SEts2-seqF和SEts2-seqR2进行PCR扩增获得935bp的产物，则认定该小鼠为SEts2基因敲除型；当由SEts2-seqF和SEts2-seqR1进行PCR扩增后获得737bp的产物，而由SEts2-seqF和SEts2-seqR2进行PCR扩增获得935bp的产物，则为杂合型，即阳性F0代小鼠。
表4 PCR反应体系
试剂 体积
2xPCR mix 10μL
SEts2-seqF 0.5μL
SEts2-seqR1/SEts2-seqR2 0.5μL
模板gDNA 1μL
超纯水 8μL
表5 PCR反应程序
Figure BDA0003450726840000051
表6小鼠基因型判定标准
Figure BDA0003450726840000052
对F0代小鼠LCR基因型鉴定的琼脂糖电泳图如图3所示，对出生的16只小鼠进行PCR及琼脂糖电泳检测，得到编号为1474、1476两只小鼠为基因编辑小鼠。对获得的两只基因编辑小鼠进行直接测序测序，对F0代小鼠进行SEts2敲除鉴定，测序结果如图4所示，编号为1474小鼠敲除长度为～166.8kb、1476小鼠敲除长度为～167kb，表明由本发明构建的gRNA1和gRNA2介导的Cas9基因敲除能够有效敲除SEts2片段。
4.F0代阳性小鼠在8周龄性成熟后与野生型C57小鼠进行配繁，出生F1代小鼠在1周龄进行剪尾鉴定。♀C57 X♂1474合笼，共出生F1小鼠21只，经基因型鉴定，共有10只阳性F1鼠。♀C57 X♂1476合笼，共出生F1小鼠11只，经基因型鉴定，共有3只阳性F1鼠。其中F1代小鼠信息如表7、表8所示。
表7 F0代与C57小鼠繁殖信息
Figure BDA0003450726840000061
表8 F1代阳性小鼠信息
品系 脚趾编号 基因型 性别 代数 出生日期
Ets2-KO 5356 杂合 ♂ F1 2018/9/9
Ets2-KO 5492 杂合 ♀ F1 2018/9/23
Ets2-KO 5611 杂合 ♀ F1 2018/10/8
Ets2-KO 5609 杂合 ♂ F1 2018/10/8
Ets2-KO 5711 杂合 ♀ F1 2018/10/15
Ets2-KO 5716 杂合 ♀ F1 2018/10/15
Ets2-KO 5717 杂合 ♂ F1 2018/10/15
Ets2-KO 5718 杂合 ♂ F1 2018/10/15
Ets2-KO 5720 杂合 ♂ F1 2018/10/15
Ets2-KO 5721 杂合 ♂ F1 2018/10/15
Ets2-KO 5796 杂合 ♀ F1 2018/10/15
上述构建SEts2敲除的动物模型的方法具有操作简单、敲除效率高的优势；由上述方法构建得到的SEts2敲除的动物模型，有助于对超级增强子参与的Ets2基因调控机制进行研究。

Claims (4)

1.基于CRISPR/Cas9构建Ets2基因超级增强子敲除动物模型的方法，其特征在于，包括如下步骤：
1)靶向小鼠SEts2序列的gRNA设计：
根据SEts2序列设计一对相应的gRNA，所述gRNA的序列如SEQ ID NO.1和SEQ ID NO.2所示；
所述靶SEts2序列位于小鼠Chr16:95721049-95979933区域；
2)gRNA制备并显微注射获得F0小鼠：
将步骤1)中的一对gRNA进行纯化，并与cas9质粒共同注射小鼠胚胎，获得F0代小鼠；
3)鉴定SEts2基因型，筛选出阳性F0代小鼠；
4)阳性F0代小鼠与野生型小鼠杂交得到F1代小鼠，F1代杂合小鼠自交获得纯合后代，从而构建得到SEts2敲除的动物模型。
2.根据权利要求1所述敲除动物模型的方法，其特征在于，所述步骤3)用于SEts2基因型鉴定的引物包括公用上游引物和两条下游引物，所述上游引物序列如SEQ ID NO.3所示，所述下游引物序列如SEQ ID NO.4和EQ ID NO.5所示。
3.一种SEts2敲除的动物模型，其特征在于，由权利要求1或2所述方法构建得到。
4.权利要求3所述动物模型可以在多个器官发育过程中研究超级增强子对Ets2基因调控的机制。
CN202111674779.1A 2021-12-31 2021-12-31 基于CRISPR/Cas9构建Ets2基因超级增强子敲除动物模型的方法及其应用 Active CN114958908B (zh)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN202111674779.1A CN114958908B (zh) 2021-12-31 2021-12-31 基于CRISPR/Cas9构建Ets2基因超级增强子敲除动物模型的方法及其应用

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN202111674779.1A CN114958908B (zh) 2021-12-31 2021-12-31 基于CRISPR/Cas9构建Ets2基因超级增强子敲除动物模型的方法及其应用

Publications (2)

Publication Number Publication Date
CN114958908A true CN114958908A (zh) 2022-08-30
CN114958908B CN114958908B (zh) 2024-03-29

Family

ID=82974904

Family Applications (1)

Application Number Title Priority Date Filing Date
CN202111674779.1A Active CN114958908B (zh) 2021-12-31 2021-12-31 基于CRISPR/Cas9构建Ets2基因超级增强子敲除动物模型的方法及其应用

Country Status (1)

Country Link
CN (1) CN114958908B (zh)

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113088521A (zh) * 2021-04-16 2021-07-09 河南省人民医院 一种基于CRISPR/Cas9技术的Ahnak2基因敲除动物模型的构建方法

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113088521A (zh) * 2021-04-16 2021-07-09 河南省人民医院 一种基于CRISPR/Cas9技术的Ahnak2基因敲除动物模型的构建方法

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
DELONG FENG等: "Chromatin organizer SATB1 controls the cell identity of CD4+ CD8+ double-positive thymocytes by compacting super-enhancers", RESEARCH SQUARE, pages 2 - 3 *

Also Published As

Publication number Publication date
CN114958908B (zh) 2024-03-29

Similar Documents

Publication Publication Date Title
Song et al. Efficient dual sgRNA-directed large gene deletion in rabbit with CRISPR/Cas9 system
Tang et al. CRISPR/Cas9-mediated gene editing in human zygotes using Cas9 protein
WO2018219093A1 (zh) 一种基于CRISPR/Cas9技术的Glrx1基因敲除动物模型的构建方法
CN107475300B (zh) Ifit3-eKO1基因敲除小鼠动物模型的构建方法和应用
CN106047930B (zh) 一种PS1基因条件性敲除flox大鼠的制备方法
Jang et al. Multiple sgRNAs with overlapping sequences enhance CRISPR/Cas9-mediated knock-in efficiency
Sakurai et al. A single blastocyst assay optimized for detecting CRISPR/Cas9 system-induced indel mutations in mice
Yan et al. Generation of multi-gene knockout rabbits using the Cas9/gRNA system
Kumagai et al. CRISPR/Cas-mediated knock-in via non-homologous end-joining in the crustacean Daphnia magna
Zhou et al. Programmable base editing of the sheep genome revealed no genome-wide off-target mutations
CN110551759B (zh) 一种提高转基因细胞重组效率的组合物及方法
Zhou et al. Generation of gene-edited sheep with a defined Booroola fecundity gene (FecBB) mutation in bone morphogenetic protein receptor type 1B (BMPR1B) via clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated (Cas) 9
Takada et al. Targeted gene deletion of miRNAs in mice by TALEN system
WO2018045727A1 (zh) 粘多糖贮积症ii型动物模型的构建方法及应用
WO2022227692A1 (zh) 一种无肌间刺鲫品系及其培育方法
Kato et al. Creation of mutant mice with megabase-sized deletions containing custom-designed breakpoints by means of the CRISPR/Cas9 system
Murakami et al. CRISPR/Cas9 nickase‐mediated efficient and seamless knock‐in of lethal genes in the medaka fish Oryzias latipes
Markossian et al. CRISPR/Cas9: a breakthrough in generating mouse models for endocrinologists
CN113699152A (zh) Slc35e2b基因敲除小鼠动物模型的构建方法和应用
Zhang et al. Generation of an oocyte-specific Cas9 transgenic mouse for genome editing
CN114958908B (zh) 基于CRISPR/Cas9构建Ets2基因超级增强子敲除动物模型的方法及其应用
CN112980881B (zh) Arvcf基因敲除动物模型的构建方法及应用
CN115261360A (zh) 一种gata6基因敲除斑马鱼模型的构建方法
CN115807037A (zh) 一种遗传可控的四倍体鱼的选育方法及三倍体鱼的制备方法
Ohtsuka Development of Pronuclear Injection-Based Targeted Transgenesis in Mice Through Cre–loxP Site-Specific Recombination

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