CN112695034A - ApoE基因缺失斑马鱼的制备 - Google Patents
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Abstract
本发明利用CRISPR/Cas9技术，构建成了ApoE基因缺失斑马鱼，具体步骤包括：1)根据ApoE基因序列，确定ApoE靶位点；2)制备gRNA；3)体外转录Cas9mRNA；4)将Cas9mRNA和gRNA混合，注射到单细胞期斑马鱼胚胎中；5)将F0胚胎饲养至性成熟；6)与野生型成鱼外交，筛选F0；7)选取可产生有效突变的F0自交，筛选F1；8)从F1代突变体中挑选相同突变的雌鱼和雄鱼，杂交得到F2代；9)筛选ApoE基因敲除纯合子即为稳定遗传的ApoE基因缺失斑马鱼。本发明构建的ApoE基因缺失斑马鱼可稳定遗传，可用于阿尔兹海默症、骨质疏松、冠心病疾病的研究。
Description
技术领域
本发明属于分子生物学领域，具体涉及ApoE基因缺失斑马鱼的制备。
背景技术
人的ApoE是一个由299个氨基酸组成的糖蛋白，在多种器官中表达，表达量最高的是肝脏，其次是大脑。大脑中表达ApoE的细胞主要是一些非神经元细胞，ApoE和Aβ相互作用，在阿尔兹海默症发病中扮演了重要角色。在周边和中枢神经系统中，神经元损伤后ApoE表达会大量增加，一些研究表明ApoE在维持神经的可塑性和功能方面十分重要。近年来，国内外学者认为ApoE是骨质疏松症的候选基因之一，ApoE基因多样性与骨量流失和骨质疏松性具有相关性。ApoE基因多态性与冠心病发生有密切关系，ApoE基因变异对冠心病的危险性有强烈而持续的影响。
斑马鱼性成熟时间短，出生后3个月就可以达到性成熟，繁殖能力强，饲养成本低，生命周期短，胚胎体外发育且透明，所需药物少，非常适宜用于阿尔兹海默症、骨质疏松、冠心病的研究。
发明内容
本发明的目的是利用CRISPR/Cas9系统构建一种ApoE基因缺失斑马鱼。
本发明的制备过程如下：
本发明靶位点DNA序列为：5’-GGATGGCGCCACCCAGAAAC-3’。
本发明提供一种基因打靶试剂盒，所述试剂盒包括两条Oligo序列，其序列为5’-GGATGGCGCCACCCAGAAAC-3’，5’-AAACGTTTCTGGGTGGCGCCAT-3’，使用该试剂盒可以用于ApoE基因表达的沉默。
本发明提供了一种基因敲除方法，所述方法的步骤如下：利用所述的Oligo片段识别目的基因的靶位点，与Cas9结合并识别靶位点处PAM序列，引导核酸酶结合到目的基因靶位点处，并开始进行剪切，形成DSB缺口，随后细胞通过非同源末端连接修复机制修复目的基因双链，造成移码突变，最终目的基因被敲除。
进一步的，所述靶点为一个或以上。
进一步的，本发明提供ApoE基因缺失斑马鱼的制备方法，所述制备方法包括：
1.根据ApoE基因序列，确定ApoE靶位点；
2.根据ApoE靶位点，设计Oligo序列；
3.构建gRNA体外转录载体；
4.PCR获得gRNA体外转录模板；
5.对步骤4获得的模板进行体外转录，得到gRNA；
6.制备Cas9 mRNA的体外转录模板；
7.体外转录Cas9 mRNA；
8.添加polyA序列、回收Cas9 mRNA；
9.将Cas9 mRNA和gRNA混合，注射到单细胞期斑马鱼胚胎中；
10.将F0胚胎饲养至性成熟；
11.与野生型成鱼外交，筛选F0；
12.选取可产生有效突变的F0自交，筛选F1；
13.从F1代突变体中挑选相同突变的雌鱼和雄鱼，杂交得到F2代；
14.筛选ApoE基因敲除纯合子即为稳定遗传的ApoE基因缺失斑马鱼。
本发明的有益效果和优点在于：
本发明构建的ApoE基因缺失斑马鱼为国内外首例。
本发明构建的ApoE基因缺失斑马鱼可稳定遗传，可用于阿尔兹海默症、骨质疏松、冠心病疾病的研究。
附图说明
图1：克隆骨架pT7-gRNA-BbsⅠ电泳图
图2：菌液PCR鉴定
图3：gRNA体外转录模板
图4：体外转录Cas9 mRNA
图5：BbeⅠ酶切
图6：ApoE基因缺失斑马鱼与野生型序列对比
具体实施方式
下面通过实施例对本发明做进一步详细说明，这些实施例仅用来说明本发明，并不限制本发明的范围。
实施例1：本发明动物模型的制备
1.实验动物
按标准化方案养殖野生型斑马鱼(TU品系)，水温28.5℃，光照/黑暗周期为14h/10h，成体斑马鱼产卵后收集胚胎，养殖于E3孵化液，以受精的小时数(hours postfertilization,hpf)或受精的天数(days post fertilization,dpf)表示胚胎和幼鱼的发育阶段。
2.CRISPR/Cas9基因敲除靶位点设计
在NCBI上查询斑马鱼ApoE基因序列，根据CRISPR/Cas9敲除原理，在http://zifit.partners.org/ZiFiT/CSquare9Nuclease.aspx上设计ApoE靶位点，靶位点包含20个碱基，靶点的选择标准为：5’-GG-(N)18-NGG-3’；其中5’端的GG二核苷酸是T7启动子的一部分，靶位点3’端是NGG。
3.构建gRNA体外转录载体
3.1用BbsⅠ酶切pT7-gRNA、切胶回收(见图1)，得到gRNA克隆骨架pT7-gRNA-BbsⅠ，酶切体系如下表1：
表1酶切体系：
3.2根据靶位点订购两条oligo，oligo1序列为5’-ATAGGATGGCGCCACCCAGAAACGT-3’，oligo2序列为5’-TAAAACGTTTCTGGGTGGCGCCTAT-3’；
3.3用ddH2O分别将oligo溶解为10μM的溶液，退火，得到粘性末端小片段，退火程序见表2；
表2退火程序
3.4将退火后的片段与回收的上述gRNA克隆骨架pT7-gRNA-BbsⅠ连接、转化，挑取克隆，用RV-M与Oligo2作为引物进行菌液PCR鉴定(58℃退火，延伸30sec，30cycle)，目的条带约为130bp(见图2)，挑取阳性克隆送去测序，选取序列正确的克隆甘油保菌、提质粒，RV-M序列：5’-AGCGGATAACAATTTCACACAGGA-3’，连接体系见表3；
表3连接体系
4.制备gRNA
4.1用T7-cr fwd和tracr rev引物对，以构建好的gRNA体外转录载体为模板，使用高保真酶PCR，得到gRNA体外转录模板(58℃退火，延伸30sec，40cycle，40μl体系)，取1μlPCR产物电泳，确认为单一条带(125bp)后直接回收PCR产物(见图3)，用于后续的实验，T7-cr fwd序列：5’-GAAATTAATACGACTCACTATA-3’，tracr rev序列：5’-AAAAAAAGCACCGACTCGGTGCCAC-3’；
4.2体外转录gRNA
gRNA体外转录体系见表4；
表4体外转录mRNA反应体系
4.3回收gRNA
4.3.1用RNase-free water将gRNA转录体系稀释到300μl，加入330μl无水乙醇；
4.3.2将溶液加到回收柱中，10000g/min离心15s；
4.3.3加入700μl的miRNA Wash SolutionⅠ，离心5-10s；
4.3.4加入500μl的Wash SolutionⅡ，离心5-10s，重复一次；
4.3.5弃去收集管中的液体，离心1min，去除残余的液体；
4.3.6加入适量95℃预热的RNase-free water，最大转速离心20-30s，收集得到gRNA溶液；
5.制备Cas9 mRNA
5.1制备Cas9 mRNA的体外转录模板：通过XbaⅠ单酶切线性化pSP6-2sNLS-spCas9载体(37℃，4h以上)；取少量电泳确认线性化完全后，直接回收线性化产物；
5.2体外转录Cas9 mRNA，mRNA体外转录体系见表5；
表5 Cas9 mRNA体外转录体系
5.3添加polyA序列，回收得到的mRNA(图4)可用于显微注射；
表6 mRNA加polyA的反应体系
6.制备F0代斑马鱼
6.1将Cas9 mRNA和gRNA混合，注射到单细胞其斑马鱼胚胎中，同时将未注射的同批胚胎作为对照，Cas9 mRNA 300-500pg，gRNA 25-200pg；
6.2取2-4dpf注射后表型正常的胚胎，提取基因组DNA，PCR、T7E1酶切检测靶位点突变效率(图5)；
6.3将未切开的条带回收、TA克隆测序，检测突变类型；
6.4选取突变效率较高、存活率较高的同批次注射的F0胚胎饲养至性成熟；
6.5与野生型成鱼外交，将3-5个1dpf的F1胚胎混和为一组提取基因组DNA；
6.6通过PCR和酶切检测靶位点突变情况；
6.7回收未切开的条带、TA克隆测序，确定突变类型；
6.8选取可产生有效突变的F0鱼交配，大量饲养F1。
7.筛选携带靶位点突变的F1成鱼
7.1将F1饲养至足够大，直至适合剪尾鳍；
7.2F1成鱼剪尾鳍、提基因组，通过PCR和酶切逐条进行基因检测，筛选出F1杂合子；
7.3回收未切开的条带、TA克隆，测序确定突变类型；
8.从F1代突变体重挑选相同突变的雌鱼和雄鱼，杂交得到F2代，放置于28.5℃培养，于4dpf取部分胚胎，每个胚胎单独提取基因组DNA，再以引物对，引物序列如下：
9.通过PCR条带分析鉴定为纯合突变的效率，进一步进行测序鉴定(图6)。
Claims (2)
1.ApoE基因缺失斑马鱼的制备，其特征在于，包括以下步骤：
1)根据ApoE基因序列，确定ApoE靶位点；
2)根据ApoE靶位点，设计Oligo序列；
3)构建gRNA体外转录载体；
4)PCR获得gRNA体外转录模板；
5)对步骤4获得的模板进行体外转录，得到gRNA；
6)制备Cas9 mRNA的体外转录模板；
7)体外转录Cas9 mRNA；
8)添加polyA序列、回收Cas9 mRNA；
9)将Cas9 mRNA和gRNA混合，注射到单细胞期斑马鱼胚胎中；
10)将F0胚胎饲养至性成熟；
11)与野生型成鱼外交，筛选F0；
12)选取可产生有效突变的F0自交，筛选F1；
13)从F1代突变体中挑选相同突变的雌鱼和雄鱼，杂交得到F2代；
14)筛选ApoE基因敲除纯合子即为稳定遗传的ApoE基因缺失斑马鱼。
2.如权利要求1所述的ApoE基因缺失斑马鱼的应用，所述的ApoE基因缺失斑马鱼用于阿尔兹海默症、骨质疏松、冠心病疾病的研究。
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