摘要
目的构建靶向CXCR7基因的CRISPR/Cas9基因编辑系统,并应用于HEK 293T细胞系。方法设计两对靶向CXCR7基因的sgRNAs,分别插入PX458载体中,并转化DH5α大肠埃希菌。经菌液PCR和测序验证,挑选序列正确的sgRNA-CXCR7-PX458质粒,转染HEK 293T细胞,用流式分选转染阳性细胞,提取其DNA,PCR扩增后测序验证。结果经测序验证,成功构建了靶向CXCR7基因的CRISPR/Cas9系统,转染HEK 293T细胞后,测序鉴定发现成功编辑CXCR7基因。结论成功构建了靶向CXCR7的sgRNA-CXCR7-PX458质粒,可在HEK 293T上成功编辑CXCR7基因,为进一步的功能研究奠定基础。
Objective To construct the CRISPR/Cas9 gene editing system targeting C-X-C chemokine receptor 7(CXCR7)gene and to edit CXCR7 gene in 293T cell line.Methods Two pairs of small guide RNAs(sgRNAs)targeting CXCR7 gene were designed and inserted into PX458 vector,which were transformed into host bacterium Escherichia coliDH5α.The correct sgRNA-CXCR7-PX458 plasmids were selected by PCR and further Sanger sequencing verification.HEK 293T cell line was transfected by DNA of sgRNA-CXCR7-PX458 plasmid.After 72 hours,GFP-positive cells were sorted by flow cytometry.We did DNA extraction of the GFP-positive cells and amplified the CXCR7 gene corresponding fragment by PCR and investigated the CXCR7 gene editing results by Sanger sequencing.Results The CRISPR/Cas9 system targeting CXCR7 gene was successfully constructed.After 293T cells were transfected,the CXCR7 gene was edited in HEK 293T cells successfully.Conclusion The sgRNA-CXCR7-PX458 plasmid targeting CXCR7 gene was successfully constructed.The CRISPR/Cas9 gene editing system targeting CXCR7 gene were used on the HEK 293T cell line,which lays a foundation for further study of BCOR function.
作者
吴洁莹
孙逊沙
李发涛
陈劲松
谢闺娥
李焱
吴韶清
WU Jieying;SUN Xunsha;LI Fatao;CHEN Jinsong;XIE Guie;LI Yan;WU Shaoqing(Guangzhou Women and Children's Medical Center,Guangzhou Medical University,Guangzhou 510623,China;Guangzhou Women and Children's Medical Center,Sun Yat-sen University,Guangzhou 510623,China;The First Affiliated Hospital,Sun Yat-sen University,Guangzhou 510080,China;KingMed School of Laboratory Medicine,Guangzhou Medical University,Guangzhou 510182,China)
出处
《广州医药》
2020年第5期16-20,共5页
Guangzhou Medical Journal
基金
国家自然科学基金项目(81601004,81102248)
广东省自然科学基金(2016A030310139,2017A030313906)
广东省科技计划项目(2014A020212019)。
