军事医学2024，Vol.48Issue(8)：586-593,8.DOI:10.7644/j.issn.1674-9960.2024.08.005
水熊虫损伤抑制基因Dsup的CRISPR/Cas9基因敲入系统构建及其对HEK 293T细胞增殖能力的影响
Construction of CRISPR/Cas9 knock-in system for tardigrade damage suppressor gene Dsup and its effect on HEK 293T cell proliferation
摘要
Abstract
Objective To construct HEK 293T cells that express tardigrade Dsup protein fused with green fluorescent protein copGFP in order to study the effect of Dsup protein on proliferation of HEK 293T cells.Methods The CRISPR/Cas9 gene knock-in system was constructed.The target gene fragments of Dsup,copGFP,EF1α and puromycin were amplified by PCR and inserted into pAAVS1-SFFV to construct the fusion vector of Dsup and copGFP,which was known as pAAVS1-SFFV-Dsup-copGFP-EF1α-Puro.pAAVS1-SFFV-Dsup-copGFP-EF1 α-Puro and pAAVS1-CRISPR-Cas9 vector were co-transfected into HEK 293T cells before Dsup gene was inserted into the AAVS1 region of HEK 293T cells via homologous recombination.The HEK 293T cells expressing Dsup gene were obtained following puromycin selection,flow cytometry sorting and genome identification.The expression of Dsup at mRNA and protein levels and proliferation-related genes(MCM2,MCM4,PCNA,Ki-67)were examined to investigate the effects of Dsup gene on the proliferation of HEK 293T-Dsup-copGFP cells.Results The pAAVS1-SFFV-Dsup-copGFP-EF1α-Puro recombinant vector was constructed,and the HEK 293T-Dsup-copGFP cells with Dsup gene inserted in the AAVS1 region were obtained,where both Dsup mRNA and protein were expressed.The cell proliferation rate of HEK 293T-Dsup-copGFP was higher than that of HEK 293T-Control-copGFP(P＜0.001).Further investigation revealed that the expressions of Ki-67 and MCM4 protein in HEK 293T-Dsup-copGFP were significantly higher than in the control group,indicating that the knock in of Dsup gene might enhance the proliferation ability of human cells by promoting the expression of Ki-67 and MCM4 protein.Conclusion A gene editing vector is constructed,and stable cell line HEK 293T-Dsup-copGFP for Dsup fusion expression with copGFP is established.The expression of Dsup gene in HEK 293T cells can promote cell proliferation,possibly by upregulating the expressions of Ki-67 and MCM4 protein.关键词
水熊虫/辐射耐受/损伤抑制蛋白/CRISPR/Cas9/细胞增殖Key words
tardigrade/radiation tolerance/damage suppressor protein/CRISPR/Cas9/cell proliferation分类
生物科学引用本文复制引用
高雨婷,张彪,贾雅丽,王海洋,范韬,习佳飞,岳文,曾泉,周军年..水熊虫损伤抑制基因Dsup的CRISPR/Cas9基因敲入系统构建及其对HEK 293T细胞增殖能力的影响[J].军事医学,2024,48(8):586-593,8.基金项目
国家自然科学基金面上项目(82273569) （82273569）
