CN108707628A - 斑马鱼notch2基因突变体的制备方法 - Google Patents
斑马鱼notch2基因突变体的制备方法 Download PDFInfo
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Abstract
本发明公开了涉及一种斑马鱼notch2基因突变体的制备方法；包括如下步骤：确定notch2基因敲除的靶点位置；以pUC19‑gRNA scaffold质粒为模板，使用引物T7‑notch2‑sfd、tracr rev进行PCR扩增；对PCR产物纯化、体外转录获得gRNA；将gRNA与Cas9蛋白显微注射入斑马鱼胚胎一细胞期中，筛选获得稳定遗传的notch2基因突变体。本发明利用CRISPR/Cas9技术，通过选择独特的一段打靶区，使得斑马鱼中的notch2基因被敲除，又不“误伤”其他基因，形成Notch2敲除的斑马鱼，为后续基因功能的深入研究提供了实验材料，对于研究Notch信号通路意义重大。
Description
技术领域
本发明涉及一种斑马鱼突变体，具体涉及一种斑马鱼notch2基因突变体的制备方法。
背景技术
CRISPR/Cas系统最早是在细菌的适应性免疫系统内发现的，其主要功能是对抗入侵的病毒及外源DNA。1987年大阪大学(Osaka University)的研究人员在Escherichiacoli K12的碱性磷酸酶基因附近发现了成簇的规律间隔的短回文重复序列(Clusteredregularly interspaced short palindromic repeat，CRISPR)和CRISPR相关基因(CRISPR-associated genes，Cas gene)，目前普遍认为有40％的细菌基因组具有这样的结构。CRISPR技术是最新出现的第三代基因组编辑工具，它能够完成RNA导向的DNA识别及编辑。CRISPR/Cas9基因编辑技术源于一种微生物防御噬菌体DNA或外源质粒入侵的后天免疫系统。CRISPR/Cas系统的防御机制可以分为三个阶段。第一个阶段称为间隔序列的获得，间隔序列被识别并被整合到CRISPR基因座中两个相邻重复单元之间。第二阶段被称为CRISPR的表达，一个主要的转录本，被RNA聚合酶从CRISPR基因座转录而来。随后，特异性的核酸内切酶将pre-crRNAs切割成小的CRISPR RNAs(crRNAs)。第三个阶段称为干扰或免疫，在crRNA和Cas蛋白形成的复合物中crRNA能识别碱基对，特别是具有完全(或几乎完全)互补的外来DNA(或RNA)的区域。这样便开始了Cas蛋白对特定位点的切割。从细菌到古细菌共有三种类型的CRISPR/Cas系统，分别是Type I、II、III，其中Type II的运用最多。Type II中包括标志性的Cas9蛋白，该蛋白主要促进crRNA的成熟，降解侵入的噬菌体DNA或者入侵的外源质粒。目前，CRISPR/Cas9技术已广泛用于小鼠、斑马鱼、果蝇、酵母、大米、小麦、细菌等各种生物上，实现了不同生物进化阶段物种的基因编辑。
与锌指核酸酶(Zinc-finger nuclease，ZFN)和转录激活样效应因子核酸酶(transcription activator-like effector nuclease，TALEN)等基因编辑技术相比，CRISPR/Cas9技术有如下优势：
1、Cas9不具有特异性；
2、gRNA体现需要敲除的基因的特异性，靶向精确，作用高效，脱靶率低；
3、廉价便捷，细胞毒性低；
4、CRISPR技术更易于操作，具有更强的可扩展性。
在人中NOTCH2受体突变会造成Alagille综合征，又称先天性肝内胆管发育不良征，是一种类及多系统的显性遗传性疾病，涉及的脏器包括肝脏、心脏、骨骼、眼睛和颜面等，肝内胆汁淤积为主要特征。面部特征主要为前额突出，眼与鼻的距离大，下颏小而尖等。目前无特殊疗法，可给予消胆胺或中药，以治疗胆汁淤积，并补充脂溶性纤维素。
在小鼠中，Notch2突变后可造成眼睛缺陷，表现为眼睛变小，明显的晶状体后的增生，形成异常的球状结构，眼睛不对称；造成心脏和血管受损，具体为心脏生长迟缓、心包水肿和出血，心肌壁变薄、心肌小梁降低；造成肾小球发育缺陷，肾脏发育缓慢。突变体小鼠最终在E16.5之前死亡。
已有报道利用TALEN技术在斑马鱼notch2第四个外显子上设计靶点并进行基因敲除，制备的notch2突变体在靶点处缺失104bp，造成移码突变，并产生终止密码子。该突变体没有表型，但可养至成鱼。而本发明用CRISPR/Cas9技术制备的不同突变类型的突变体notch2-/-均未发现明显的表型，并且在notch2不同突变类型的F2中均未筛选到纯合子斑马鱼。经χ2检验，F2中杂合子和野生型斑马鱼的数量符合2∶1的比例，故确定notch2存在纯和致死现象，并初步统计纯合子的最大存活时间约为16dpf。这说明由于作用机制、作用靶点等不同，运用不同的基因编辑方法制备的突变体在一定程度上存在着差异。
发明内容
本发明的目的在于提供一种斑马鱼notch2基因突变体的制备方法。
本发明的目的是通过以下技术方案来实现的：
本发明涉及一种斑马鱼notch2基因突变体的制备方法，所述方法包括如下步骤：
S1、确定notch2基因敲除的靶点在斑马鱼notch2的基因序列的第四个外显子上；
S2、根据步骤S1确定的靶点序列设计扩增引物；
S3、以pUC19-gRNA scaffold质粒为模板，使用引物T7-notch2-sfd、tracr rev进行PCR扩增；
S4、对步骤S3的PCR产物进行纯化，体外转录获得gRNA；
S5、将gRNA与Cas9蛋白导入斑马鱼中；
S6、培养获得稳定遗传的斑马鱼notch2基因突变体。
优选的，步骤S2中，所述靶点序列为GGCGTTTGTGTGAACACCAT(SEQ ID NO.2)。
优选的，步骤S3中，pUC19-gRNA scaffold质粒模板序列为：GTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTTT(SEQ IDNO.1)。
优选的，步骤S3中，所述引物T7-notch2-sfd的序列为TAATACGACTCACTATAGGCGTTTGTGTGAACACCATGTTTTAGAGCTAGAAATAGC(SEQ ID NO.3)。
优选的，步骤S3中，所述引物tracr rev的序列为AAAAAAAGCACCGACTCGGT GCCAC(SEQ ID NO.4)。
优选的，步骤S4中，所述gRNA的序列为TAATACGACTCACTATAGGCGTTTGTGTGAACACCATGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTTT(SEQ ID NO.7)。
优选的，步骤S5中，将gRNA与Cas9蛋白导入斑马鱼具体为：将gRNA与Cas9蛋白混合，显微注射到斑马鱼一细胞期胚胎中；其中，gRNA终浓度为100ng/μL，Cas9蛋白终浓度为800ng/μL。
优选的，步骤S6具体包括如下步骤：
A1、对导入gRNA与Cas9蛋白的斑马鱼进行notch2基因敲除检测，确定notch2 F0靶点突变效率；
A2、将notch2 F0基因检测敲除成功的成鱼与野生型斑马鱼外交，得到F1胚胎；经基因型鉴定获得notch2 F1突变体斑马鱼；
A3、将相同突变的notch2 F1突变体斑马鱼内交，获得notch2 F2突变体斑马鱼；
A4、鉴定为F2中notch2基因敲除的纯合子即所述稳定遗传的斑马鱼notch2基因突变体。
优选的，步骤A1中，notch2基因敲除检测采用的引物序列为F：CCCATGTGCCAACAAGGGTA(SEQ ID NO.5)和R：ACAAGTCTAGCTTCTCTTCAGATT(SEQ ID NO.6)。
本发明选取的斑马鱼notch2基因，是Notch受体之一，在发育上起重要作用。在人类中NOTCH2基因突变会造成Alagille综合征，又称先天性肝内胆管发育不良征，患者会出现前额突出，眼与鼻的距离大，下颏小而尖等面部特征；在小鼠中，Notch2基因突变会导致肾脏发育迟缓，眼睛变小，心血管异常等，最终在E16.5之前死亡。斑马鱼notch2基因位于斑马鱼8号染色体上，全长10095bp，编码2475个氨基酸，含有34个外显子和33个内含子。本发明根据T7启动子的靶位点选择规律，选取了比较靠前的、位于Notch2基因第4个外显子上的靶点，F0敲除效率超过70％。制备了8种突变类型，其中-4、-11和+26bp三种类型的突变体分别形成了256、250和212个氨基酸的截短蛋白。
本发明利用CRISPR/Cas9制备的-4、-11和+26bp三种不同类型的突变体notch2-/-均未发现明显的表型，并且在notch2不同突变类型的F2中均未筛选到纯合子斑马鱼。经χ2检验，F2中杂合子和野生型斑马鱼的数量符合2∶1的比例，故确定notch2存在纯和致死现象，并初步统计纯合子的最大存活时间约为16dpf。这说明本发明靶点特异性较高，未出现脱靶现象，制备的突变体为今后研究相应的疾病提供了可靠的材料保障。
与现有技术相比，本发明具有如下有益效果：
1、首次在斑马鱼中利用CRISPR/Cas9技术获得notch2突变体；
2、notch2突变体可稳定遗传，方便对notch2功能的深入研究；
3、notch2-/-突变体出现早期纯合致死现象；
4、利用CRISPR/Cas9技术，设计独特的一段打靶区，使得斑马鱼中的notch2基因被敲除，又不“误伤”其他基因，形成Notch2敲除的斑马鱼。
附图说明
图1为notch2基因F0敲除检测示意图；其中，a为notch2 F0斑马鱼胚胎PCR产物，b为T7E1内切酶酶切鉴定结果，c为PCR产物测序结果；
图2为notch2 F0 germline transmission检测结果；
图3为notch2 F1成年斑马鱼基因型检测结果。
具体实施方式
下面结合实施例对本发明进行详细说明。以下实施例将有助于本领域的技术人员进一步理解本发明，但不以任何形式限制本发明。应当指出的是，对本领域的普通技术人员来说，在不脱离本发明构思的前提下，还可以做出若干调整和改进。这些都属于本发明的保护范围。
实施例
1材料及设备
1.1实验用鱼
本实验中所用的斑马鱼均为AB品系，购买于中国科学院上海生命科学研究院生物化学与细胞生物学研究所斑马鱼平台。
1.2质粒
pUC19-gRNA scaffold质粒来源于文献：Chang N，Sun C，Gao L，Zhu D，Xu X，ZhuX，Xiong JW，Xi JJ.Genome editing with RNA-guided Cas9 nuclease in zebrafishembryos，Cell Res，2013，23(4)：465-472。
在gRNA产物合成中用到的pUC19-gRNA scaffold质粒模板序列为：
GTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTTT(SEQ ID NO.1)。
1.3主要试剂
DNA Clean&Contentrator-5(ZYMO RESEARCH，D4004)，普通DNA纯化试剂盒(TIANGEN，DP204-03)，T7 in vitro Transcription Kit(Ambion，AM1314)，乙醇(无水乙醇)(国药集团化学试剂有限公司，10009218)，GenCrispr NLS-Cas9-NLS(金斯瑞，Z03389-25)，Premix TaqTM(Ex TaqTM Version 2.0plus dye)(TAKARA，RR902)，DNAMarker I(TIANGEN，MD101-02)，T7endonuclease 1(NEW ENGLAND Inc.，M0302L)，快速质粒小提试剂盒(TIANGEN，DP105)，DH5α感受态细胞(TIANGEN，CB101-03)，2BEasyTaqPCR SuperMix(+dye)(TAKARA，AS111-12)，LB Broth(上海生工，D915KA6602)，LB Brothagar(上海生工，D911KA6566)，pMDTM19-T Vector Cloning Kit(TAKARA，6013)。
1.4主要仪器
PCR仪(品牌：BIO-RAD，型号：c1000 TouchTMThermal Cycler)，小离心机(品牌：eppendorf，型号：Centrifuge 5424)，震荡混匀仪(品牌：VORTEX-GENIE，型号：G560E)，紫外分光光度计(品牌：Thermo Scientific，型号：Nanodrop 2000C)，电泳仪(品牌：BIO-RAD，型号：PowerPac Basic)，照胶仪(品牌：BIO-RAD，型号：Gel Doc EZ Imager)，电子天平(品牌：METTLER TOLEDO，型号：AL104)，玻璃毛细管(品牌：WPI，型号：TW100F-4)，Milli-Q Direct8超纯水系统(品牌：Millipore，型号：Milli-Q Direct 8)，垂直拉针仪(品牌：NARISHIGE，型号：PC-10)，恒温摇床(品牌：Innova，型号：40R)，磨针器(品牌：NARISHIGE，型号：EG-400)，微量注射泵(品牌：WARNER，型号：PLI-100A)，恒温水浴锅(品牌：精宏，型号：H1401438，DK-8D)，4℃冰箱(品牌：Haier，型号：HYC-610)，-40℃低温冰箱(品牌：Haier，型号：DW-40L508)，-80℃超低温冰箱(品牌：Pana-sonic，型号：MDF-U53V)，高压蒸汽灭菌锅(品牌：SANYO，型号：MLS-3780)。
2实验方法
2.1 gRNA合成
(1)靶点设计
a、下载序列：在Ensembl数据库查找并下载斑马鱼notch2的基因序列。
b、靶点设计：利用http：//zifit.partners.org/ZiFiT/ChoiceMenu.aspx网站在notch2基因ATG之后的外显子序列上设计靶点(表1)。notch2设计靶点在第四个外显子上。
c、靶点特异性检测：在NCBI网站将设计的靶点序列通过blast比对，验证靶位点特异性。
d、亲本检测：将用于基因敲除的野生型斑马鱼剪尾并用碱裂解法获得基因组DNA，进行PCR扩增靶点附近的一段序列。
e、酶切检测：用T7E1内切酶酶切检测野生型斑马鱼，看T7E1酶能否将扩增的片段切开，若切不开，则可用于后续敲除检测；若被切开，则需要根据扩增序列信息选择特异性的酶进行酶切检测。
f、测序鉴定：将PCR产物送测序，峰图及序列比对，确认亲本为纯合子，不存在自然突变，从而保证后续制备的突变体为基因敲除后造成的。
表1 notch2靶位点序列
(2)设计检测引物：设计的引物应保证距离靶点两侧大于100bp，并且上下游引物到靶点的距离与下游引物到靶点的距离应相差大于100bp，至少50bp。引物扩增应具备特异性，扩增片段约500bp。引物在上海生工生物工程股份有限公司合成(表2)。
表2实验所用引物信息
(3)gRNA产物合成：以pUC19-gRNA scaffold质粒为模板，使用引物T7-notch2-sfd、tracr rev和2×EasyTaq PCR Super Mix(+dye)扩增片段并用试剂盒纯化。
(4)体外转录：
表3反应体系
| Nuclease-free Water | to 20μL |
| DNA template | 1μg |
| 10×Transcription Buffer | 2μL |
| 10mM ATP | 1μL |
| 10mM CTP | 1μL |
| 10mM GTP | 1μL |
| 10mM UTP | 1μL |
| T7Enzyme Mix | 2μL |
注意：最后添加10×Transcription Buffer和T7Enzyme mix
混匀并短暂离心后，37℃孵育80min；之后向体系中加入1μL TURBO DNase并混匀，短暂离心后37℃孵育15min。
(5)纯化gRNA：
a、向20μL体外转录体系中加入2.5μL 4M的LiCl和100μL无水乙醇，混匀并短暂离心后放于-80℃冰箱至少1h。
b、到时间后从冰箱取出，4℃，12000rmp，离心15min。弃上清后用70％乙醇清洗沉淀。4℃，8000rmp，离心5min。弃上清后将离心管放于通风橱中使乙醇挥发干净。
c、根据沉淀大小加入适量DEPC水溶解gRNA沉淀。
d、用Nanodrop检测浓度和OD值并用电泳检测。
所述gRNA的序列为TAATACGACTCACTATAGGCATCTGCATGAATACACAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTTT(SEQ IDNO.7)。
2.2显微注射
将gRNA与Cas9蛋白(购买于南京金斯瑞生物科技有限公司[GenCrispr NLS-Cas9-NLS(金斯瑞，Z03389-25)])混合，利用显微注射仪器将混合后的物质注射到斑马鱼一细胞期胚胎中，每次注射都留一批未注射的同批次胚胎作为对照组。混合注射终浓度：gRNA为100ng/μL，Cas9蛋白为800ng/μL。
2.3检测敲除效率
a、提取鱼卵基因组
每组5枚卵，加35μL 50mM NaOH，95℃孵育20min，中间取出振荡，短暂离心一次。之后加3.5μL 1M的Tris.HC1(pH≈8.0)，剧烈振荡混匀后离心。
b、PCR扩增目的片段
根据靶点附近设计的引物F：CCCATGTGCCAACAAGGGTA(SEQ ID NO.5)和R：ACAAGTCTAGCTTCTCTTCAGATT(SEQ ID NO.6)扩增目的片段。
表4 PCR反应体系
| H2O | to 25μL |
| 酶 | 12.5μL |
| F | 0.5μL |
| R | 0.5μL |
| Template | 10ng |
PCR反应条件：
98℃预变性2sec；98℃变性10sec，58℃退火30sec，72℃延伸1min，共34个循环；72℃再延伸5min；4℃保存。
2％琼脂糖凝胶120V电泳27min。
c、T7E1内切酶酶切检测
表5
| H2O | to 10μL |
| PCR产物 | 5μL |
| Buffer | 1.1μL |
95℃孵育5min，冷却至室温，加0.25μL T7E1酶，37℃孵育45min。
d、电泳检测
电泳后利用凝胶电泳成像仪对电泳的琼脂糖凝胶成像，观察目的条带，判断敲除是否成功。
3实验结果
3.1 notch2突变体的构建
3.1.1 notch2 F0基因敲除检测结果
T7E1酶切结果显示notch2基因敲除成功。利用Image Lab 5.1软件计算敲除效率达到70％以上。测序峰图显示在20bp靶点处出现套峰，证明敲除成功(图1)。
3.1.2 notch2 F0 germline transmission检测结果
取23尾notch2 F0基因检测敲除成功的成鱼与野生型斑马鱼外交，得到的F1胚胎5枚一管，取3管进行T7E1酶切鉴定，酶切结果显示，有12尾斑马鱼将突变传递给后代(图2)，其中雌鱼9尾，雄鱼3尾。
3.1.3 notch2 F1突变体斑马鱼检测
剪尾检测51尾notch2(外交)斑马鱼，经T7E1检测到47尾阳性斑马鱼，进行TA克隆，其中有17尾发生有效突变，氨基酸序列发生移码突变并提前产生终止密码子。
发生有效突变的斑马鱼中+26bp的突变体只得到1尾雄鱼；-11bp的突变体有两种类型，一种得到5尾突变体，2雌3雄，另一种得到2尾突变体，1雌1雄；-10bp和-2bp的突变体各得到1尾雌鱼；-4bp的突变体仅筛选到1尾雄鱼；-1bp的的突变体得到6尾，其中有2尾突变基因型完全相同的，并且1雌1雄，另外-1bp的2尾突变体还有一个突变碱基，但均为雄鱼(图3)。
3.1.4 notch2突变体斑马鱼形态学观察
将相同突变类型的notch2+/-斑马鱼内交后，没有观察到notch2-/-突变体明显的表型。在notch2不同突变类型的F2中均未筛选到纯合子斑马鱼。经χ2检验，F2中杂合子和野生型斑马鱼的数量符合2∶1的比例，故确定notch2存在纯和致死现象，并初步统计纯合子的最大存活时间约为16dpf。
综上所述，本发明首次在斑马鱼中利用CRISPR/Cas9技术获得notch2突变体。作为首例利用CRISPR/Cas9技术敲除的Notch2基因模式动物斑马鱼，可以排除人为因素干预，对于Notch2基因的功能研究意义重大，同时与传统基因敲除的技术相比，周期短，使得Notch2基因更快的被敲除。虽然已有报道用TALEN技术在斑马鱼notch2第四个外显子上设计靶点并进行基因敲除，但制备的notch2el517突变体没有表型，并可养至成鱼。考虑到notch2基因对机体的重要作用，为深入研究基因的具体功能，我们首次在斑马鱼的上利用CRISPR/Cas9技术制备notch2突变体。本发明利用CRISPR/Cas9技术所制备的突变体与TALEN技术制备的突变体不仅制备突变体的方法不同，并且突变体在个体水平上存在意想不到的较大差异，即我们用CRISPR/Cas9技术制备的notch2突变体出现早期纯合致死现象。这说明由于作用机制、作用靶点等不同，运用不同的基因编辑方法制备的突变体在一定程度上存在着差异。制备的notch2突变体为后续基因功能的深入研究提供了实验材料，对于研究Notch信号通路意义重大。
序列表
<110> 上海海洋大学
<120> 斑马鱼notch2基因突变体的制备方法
<130> 2018
<141> 2018-05-28
<160> 7
<170> SIPOSequenceListing 1.0
<210> 1
<211> 83
<212> DNA
<213> pUC19-gRNA scaffold质粒()
<400> 1
gttttagagc tagaaatagc aagttaaaat aaggctagtc cgttatcaac ttgaaaaagt 60
ggcaccgagt cggtgctttt ttt 83
<210> 2
<211> 20
<212> DNA
<213> Danio rerio
<400> 2
ggcgtttgtg tgaacaccat 20
<210> 3
<211> 57
<212> DNA
<213> Artificial sequence
<220>
Claims (9)
1.一种斑马鱼notch2基因突变体的制备方法，其特征在于，所述方法包括如下步骤：
S1、确定notch2基因敲除的靶点在斑马鱼notch2基因序列的第四个外显子上；
S2、根据步骤S1确定的靶点序列设计扩增引物；
S3、以pUC 19-gRNA scaffold质粒为模板，使用引物T7-notch2-sfd、tracr rev进行PCR扩增；
S4、对步骤S3的PCR产物进行纯化，体外转录获得gRNA；
S5、将gRNA与Cas9蛋白导入斑马鱼中；
S6、培养获得稳定遗传的斑马鱼notch2基因突变体。
2.根据权利要求1所述的斑马鱼notch2基因突变体的制备方法，其特征在于，步骤S2中，所述靶点序列为如SEQ ID NO.2所示的序列。
3.根据权利要求1所述的斑马鱼notch2基因突变体的制备方法，其特征在于，步骤S3中，pUC19-gRNA scaffold质粒模板序列为如SEQ ID NO.1所示的序列。
4.根据权利要求1所述的斑马鱼notch2基因突变体的制备方法，其特征在于，步骤S3中，所述引物T7-notch2-sfd的序列为如SEQ ID NO.3所示的序列。
5.根据权利要求1所述的斑马鱼notch2基因突变体的制备方法，其特征在于，步骤S3中，所述引物tracr rev的序列为如SEQ ID NO.4所示的序列。
6.根据权利要求1所述的斑马鱼notch2基因突变体的制备方法，其特征在于，步骤S4中，所述gRNA的序列为如SEQ ID NO.7所示的序列。
7.根据权利要求1所述的斑马鱼notch2基因突变体的制备方法，其特征在于，步骤S5中，将gRNA与Cas9蛋白导入斑马鱼具体为：将gRNA与Cas9蛋白混合，显微注射到斑马鱼一细胞期胚胎中；其中，gRNA终浓度为100g/μL，Cas9蛋白终浓度为800ng/μL。
8.根据权利要求1所述的斑马鱼notch2基因突变体的制备方法，其特征在于，步骤S6具体包括如下步骤：
A1、对导入gRNA与Cas9蛋白的斑马鱼48hpf后进行notch2基因敲除检测，确定notch2 Fo靶点突变效率；
A2、将notch2 F0基因检测敲除成功的成鱼与野生型斑马鱼外交，得到F1胚胎；经基因型鉴定获得notch2 F1突变体斑马鱼；
A3、将相同突变的notch2 F1突变体斑马鱼成鱼内交，获得notch2 F2突变体斑马鱼；
A4、鉴定为F2中notch2基因敲除的纯合子即所述稳定遗传的斑马鱼notch2基因突变体。
9.根据权利要求8所述的斑马鱼notch2基因突变体的制备方法，其特征在于，步骤A1中，notch2基因敲除检测采用的引物序列为如SEQ ID NO.5和SEQ ID NO.6所示的序列。
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