靶向敲除猪ApoE基因的CRISPR/Cas9系统构建及基因组检测

Construction of CRISPR/Cas9 System Targeting Porcine ApoE Gene and Detection in Genome

通过敲除猪的ApoE基因,为后期构建ApoE缺陷型动物模型奠定基础。利用CRISPR/Cas9系统和实验室前期构建的报告载体,分别构建了靶向猪ApoE基因的两个CRISPR/Cas9表达载体和相应的报告载体。通过CRISPR/Cas9表达载体和RG(Red-GFP)报告载体转染HEK 293T细胞荧光观察结果显示,构建的CRISPR/Cas9表达载体均有较高的活性,进一步在流式细胞仪上检测其活性分别为6.50%和6.83%。将CRISPR/Cas9表达载体和RPG(Red-PuroR-GFP)报告载体转染PK15细胞系,经过嘌呤霉素药物筛选后,对富集到的细胞进行基因组检测。结果显示本系统能够对猪PK15细胞中的ApoE基因进行有效的敲除,其敲除效率分别为40%、53.3%。试验结果可为ApoE敲除猪动物模型的构建提供理论依据。

To construct Apo E knockout animal model later by disrupting porcine Apo E gene, two CRISPR/Cas9 expressing vectors targeting Apo E gene and corresponding surrogate reporter vectors were constructed. Firstly, we verified the activity of these CRISPR/Cas9 expressing vectors in HEK293T cells using RG （Red-GFP） surrogate reporter vector. The observed results under fluorescence microscope demonstrated that the CRISPR/Cas9 expressing vectors worked well, and their functional efficiency detected by Flow Cytometer were 6.50% and 6.83%, respectively. Then, the PK15 cells were transfected with CRISPR/Cas9 expressing vectors together with RPG （Red-Puro-GFP） surrogate reporter vectors, and puromycin treatment was performed to enrich the cells in which our CRISPR/Cas9 system successfully cleaved its targets. Mutations in the target loci of the enriched cells were verified. The result indicated that porcine Apo E gene could be effectively deleted using our CRISPR/Cas9 expressing vectors, and their functional efficiency could reach 40% and 53.3%, respectively. This research would provide theoretical basis for the construction of Apo E knockout pig animal model.