CRISPR/Cas9介导的猪Six1和Six4基因敲除细胞系的建立

Establishment of Six1 and Six4-deficient pig cell lines via CRISPR/Cas9 targeting

目的:利用干细胞囊胚互补技术再生同种或异种动物器官已得到验证,其重要的前提是需要构建出相应的器官缺失动物模型。本文拟通过CRISPR/Cas9基因编辑技术建立猪Six1单基因和Six1/Six4双基因敲除猪细胞系,为构建肾脏发育缺陷和缺失猪模型奠定重要的研究基础。方法:选取猪Six1基因和Six4基因第一外显子为敲除靶点,分别设计并合成单导向RNA(single guide RNA,sg RNA),将其插入含有Cas9骨架的PX330质粒,分别构建Six1基因和Six4基因敲除打靶载体PX330-sg RNA1和PX330-sg RNA4;用T7EN1酶验证打靶载体敲除效率后,将高效打靶载体与含G418抗性的质粒(p CMV-td Tomato)共转染原代猪胎儿成纤维细胞(porcine fetal fibroblasts,PFFs)中,并通过药物筛选获得单克隆细胞群落,随后对单克隆细胞进行基因型鉴定。结果:分别成功构建Six1和Six4基因的Cas9/sg RNA表达载体。转染Six1基因打靶载体经药物筛选后,利用PCR方法鉴定所获得的Six1基因敲除单克隆细胞系48个,其中Six1-/-细胞系21个。通过同样方法同时转染Six1基因和Six4基因打靶载体,经药物筛选后共获得44个单克隆细胞系,其中Six1-/-Six4-/-细胞系为13个。结论:通过CRISPR/Cas9基因编辑技术可高效获得Six1单基因敲除细胞系和Six1/Six4双基因敲除细胞系,为构建肾脏发育缺陷猪动物模型提供了基础,并有助于研究Six1和Six4基因在猪肾脏发育中的功能。

Objective：The concept of animal organs regeneration via stem cells injection and blastocyst complementation has been proved. To apply this principle for organ regeneration,the specific organ-deficient large animals must be created firstly in order to make the specific organ‘developmental niche＇. In this study,we attempt to generate the Six1-deficient and Six1/Six4-deficient pig cell lines by CRISPR/Cas9,in order to construct pig kidney-deficient model next step. Methods：The first exon of Six1 and Six4 gene was chosen for the target to design the sg RNA and the two sg RNAs were cloned into PX330 plasmids containing Cas9 skeleton respectively.The efficiency of PX330-sg RNA vector was tested by T7 EN1 enzyme assay analysis,then,the efficient PX330-sgRNA was co-transfected with the G418 resistant plasmid（p CMV-td Tomato）into the primary porcine fetal fibroblasts（PFFs）. The monoclonal cell populations were obtained through the G418 screening and the genotypes of monoclonal cells were identified by sequencing analysis.Results：Cas9/sgRNA expression vectors for Six1 and Six4 gene targeting were constructed successfully. Total 48 monoclonal cell populations with presumed Six1 gene mutation were obtained and identified by PCR,among them 21 cell colonies showed Six1 bialellic mutation（Six1-/）. By the same way,we obtained total 44 monoclonal cell populations for both Six1 and Six4 targeting and 13 of them showed Six1^-/-Six4^-/-genotypes. Conclusion：The Six1^-/-and Six1^-/-Six4^-/-cell lines were obtained by highly efficient CRISPR/Cas9 targeting,which can provide the possibility in production of the pig kidney-deficient model and also be helpful for the function investigation of Six1 and Six4 genes in pig kidney development.