利用CRISPR/Cas9n系统构建Asxl2基因敲除的NIH3T3稳定细胞系

摘要： 目的 利用 CRISPR/Cas9n 系统在 NIH3T3 小鼠胚胎成纤维细胞系中敲除 Asxl2 基因。方法 设计一对靶 向小鼠 Asxl2 基因第 5 个外显子的小向导 RNA （sgRNA）， 分别克隆进 pX462 载体。将测序鉴定正确的重组质粒转染 至 NIH3T3 细胞中， 利用有限稀释法得到单细胞， 通过培养获得单克隆细胞系。提取单克隆细胞系基因组 DNA， ge? notyping PCR 扩增出靶位点附近的 DNA 片段并测序。利用 Western blot 方法检测细胞株中 Asxl2 的敲除效果。结 果 成功构建靶向 Asxl2 的 CRISPR/Cas9n 重组质粒。将 2 个重组质粒共转染 NIH3T3 细胞， 嘌呤霉素筛选后得到亚 克隆细胞系， 并且经 genotyping PCR 测序验证得到一株正确的单克隆细胞系。Western blot 证实敲除 Asxl2 后， 该 NIH3T3 细胞系中 Asxl2 蛋白表达缺失。结论 通过这个系统得到了靶向 Asxl2 的 CRISPR/Cas9n 重组质粒及稳定 敲除 Asxl2 的NIH3T3 细胞系。

Construction of Asxl2 gene knock out stable NIH3T3 cell line with CRISPR/Cas9n system

Abstract：Objective To knock out Asxl2 gene in murine embryonic fibroblast cell line NIH3T3 using CRISPR/Cas9n system. Methods A pair of sgRNAs which targeted exon 5 of Asxl2 gene were designed and subcloned into the pX462 vec? tor. The recombined plasmids were verified by sequencing and transfected into NIH3T3 cell line. Single cells were isolated through serial dilutions, followed by an expansion period to obtain new monoclonal cell lines. The genomic DNA of the new monoclonal cell lines was extracted and a DNA fragment flanked the target site was amplified by genotyping PCR then se? quenced. Lastly, western blotting were applied to confirm whether Asxl2 was successfully knocked out. Results The CRIS? PR/Cas9n plasmids that targeted Asxl2 were successfully constructed. NIH3T3 cells were co-transfected with the two recom? binant constructs. After puromycin selection, subclonal cell lines were obtained and one of them was validated by genotyping PCR-sequencing. Western blotting also confirmed that Asxl2 was completely depleted in the NIH3T3 cell line. Conclu? sion CRISPR/Cas9n plasmids that targeted Asxl2 were successfully constructed therefore a Asxl2 knockout NIH3T3 stable cell line was established via this system.