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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