摘要
目的:通过CRISPR/Cas9基因编辑技术和流式细胞术相结合,获得可编辑Plac9基因敲除的人胚肾细胞株(293T).方法:根据Plac9外显子设计了4个sgRNAs(S1,S2,S3,S4),分别将其与PX458载体连接,构建了PX458-S1(S2/S3/S4)载体.通过转染试剂lipo2000将表达载体分别转染至293T细胞中,并以流式细胞术对带绿色荧光蛋白标记的细胞进行单细胞分选,分选后的细胞培养一段时间后,用基因组测序和错配酶酶切进行筛选.从筛选好的细胞中提取蛋白,进行WesternBlot检测敲除效率.结果:采用CRISPR/Cas9和流式细胞术结合技术成功构建了Plac9蛋白表达缺失的人胚肾细胞株.结论:该方法简便快捷、效率高,可广泛地用于编辑各种细胞和细胞功能研究.
Objective:To obtain editable Plac9 gene knockout 293T cell line through CRISPR/Cas9 gene editing and flow cytometry.Methods:4 sgRNAs(S1,S2,S3,S4)were designed and respectively connected with PX458 carrier,according to the exons of Plac9 gene,then vectors PX458-S1(S2/S3/S4)were constructed.293T cells were transfected with the expression vectors by transfection reagent lipo2000.The green fluorescent protein labeled cells were then concentrated via single cell sorting by flow cytometry.Selected cells were cultured and identified with genomic sequencing and mismatched enzyme digestion.The protein was extracted from the selected cells and the knockout efficiency was measured by Western Blot.Results:Protein Plac9 knockout 293T cell line was successfully constructed by employing CRISPR/Cas9 and flow cytometry technology.Conclusion:This method is convenient and efficient,which can be widely applied for editing any type of cell lines and for the study of cell functions.
作者
薛璐
欧阳聪
孙宁远
秦绪慧
XUE Lu;OUYANG Cong;SUN Ningyuan;QIN Xuhui(Institute for Medical Biology and Hubei Provincial Key Laboratory for Protection and Application of Special Plants in Wuling Area of China,College of Life Sciences,South-Central University for Nationalities,Wuhan 430074,China)
出处
《中南民族大学学报（自然科学版）》
CAS
2019年第2期189-192,共4页
Journal of South-Central University for Nationalities：Natural Science Edition
基金
国家自然科学基金资助项目(31101047)
