通过CRISPR/Cas9和流式细胞术建立敲除PLAC9基因的人胚肾细胞株
Establishment of hPLAC9 gene knockout 293T cell line via CRISPR/Cas9 and flow cytometry technologies
投稿时间:2017-08-29  修订日期:2017-10-11
DOI:
中文关键词: CRISPR/Cas9  基因敲除  PLAC9
英文关键词: CRISPR/Cas9  gene knockout  PLAC9
基金项目:国家自然科学基金项目
作者单位E-mail
薛璐 生物医学研究所 sparker830305@hotmail.com 
欧阳聪 生物医学研究所 1505527764@qq.com 
秦绪慧 生物医学研究所  
孙宁远 生物医学研究所  
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中文摘要:
      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hPLAC9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PX458-S1(S2/S3/S4)载体。通过转染试剂lipo2000将表达载体分别转染到293T细胞中,再通过流式细胞术对带绿色荧光的细胞进行单细胞分选。将分选后的细胞培养一段时间,通过基因组测序及错配酶酶切进行筛选。将筛选好的细胞提取蛋白,做Western Blot检测敲除效率。结果:我们成功地构建了plac9蛋白表达缺失的人胚肾细胞株,可用于下一步的功能研究。
英文摘要:
      Objective:Obtain hPLAC9 gene knockout 293T cell line through CRISPR/Cas9 technology and flow cytometry. Methods: 4 sgRNAs (S1,S2,S3,S4) were designed according to the exons sequences of hPLAC9 gene,thenparticular vectors (PX458-S1/S2/S3/S4) were constructed.293T cells were transfected with lipo2000 then the green fluorescent proteinlabeled cells were concentrated via single cell sorting in flow cytometry.Selected cells were cultured andidentified with genomic sequencing and enzyme digestion.The knockout efficiency was caculated through Western blotting.Results:We successfully established hplac9 knockout 293T cell line for futher functional study.
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