基于CRISPR/Cas9系统制备猪Pax2基因敲除细胞系

Establishment of Pax2 Gene-Knockout Pig Cell Lines Based on CRISPR/Cas9 System

肾脏发育缺陷或缺失的猪模型可为人源化肾脏器官在异种动物体内再生提供“发育空位”,因此敲除肾脏发育关键基因Pax2(Paired box2)获得该动物模型是肾脏再生的关键步骤。本文利用生物信息学分析软件比对分析了不同物种间Pax2蛋白结构,明确了猪Pax2蛋白在氨基酸序列、二级结构和三级结构方面与人、小鼠具有相似性。结合Pax2蛋白在人和小鼠肾脏发育中的功能,选择猪Pax2基因第8外显子序列作为打靶目标区间,设计并构建了用于敲除猪Pax2基因的CRISPR/Cas9打靶载体。在检测CRISPR/Cas9载体打靶猪Pax2基因效率的基础上,利用细胞核转染和G418药物筛选获得巴马小型猪细胞单克隆。通过PCR、T载体克隆和Sanger测序检测各细胞单克隆Pax2基因的敲除情况,鉴定出33个双等位基因敲除Pax2基因的巴马小型猪细胞系,敲除效率达到45.21%(33/73)。本研究为肾脏缺失或肾发育不全的猪模型的获得奠定实验基础,同时为后续在猪体内再造人源化肾脏以及肾发育疾病的研究提供了研究材料。

The pig model with kidney deficiency or absence might provide a“development niche”for humanized kidney regeneration in the xenogeneic animals.The key kidney development gene Pax2 deletion is the crucial step for kidney vacancy model generation.The Pax2 protein structures of human,mouse and porcine were compared and analyzed via the bioinformatics software.The results of bioinformatics confirmed that amino acid sequence,secondary structure,and tertiary structure of Pax2 protein of porcine were similar with those of mouse and human.Combined with the functions of human and mouse Pax2 proteins in kidney development,the eighth exon of porcine Pax2 gene was selected as the target region.The two CRISPR/Cas9 vectors for knocking-out porcine Pax2 gene were designed and constructed.Based on testing the target efficiency of the CRISPR/Cas9 vectors,the Bama-pig cell colonies were obtained via the nucleofection and G418 drug screening.The Pax2 sequences of these colonies were detected by PCR,T-vector cloning and Sanger sequencing.As a results,33 colonies among them had been identified as biallelic deletions.The efficiency was 45.21%(33/73).This study provides the foundational information for the kidney deficiency or absence model establishments,and the materials for the following humanized kidney regeneration and the renal development diseases research.