生物技术通报Issue(8)：219-224,6.DOI:10.13560/j.cnki.biotech.bull.1985.2015.08.032
建立CRISPR/Cas9慢病毒系统高效敲除人源AIP1基因
Establishment of a CRISPR/Cas9 Lentiviral System for Knockout Gene AIP1 of Human
摘要
Abstract
This work aims to establish the CRISPR/Cas9 system of knocking out geneAIP1for producing nephocyte cell(293T)of human embryo in which geneAIP1 efficiently, stably and permanently is knocked out. Three 20 bp sgRNAs(sp1, sp2 and sp3)targeting AIP1 exons were designed and inserted in PX458 vector to construct PX458-sgRNA expression vector for knockout. CRISPR/Cas9 efficiency of knockout was assessed using T7E1 assay. sgRNA of the maximum knockout efficiency was inserted into lentiCRISPRv2 vector to construct lentiCRISPRv2-sgRNA expression vector. The correct recombinant plasmid was transfected into 293T cells. The supernatant was collected and filtered, and then infected the 293T cells. The stable 293T cell lines were generated by limiting diluting the cells in which genesAIP1 were knocked out successfully. The expression level ofAIP1 in the stable 293T cells were detected by Western blot. Three sgRNAs of AIP1 were correctly inserted into PX458 vector respectively, T7E1 verified that the knockout rate of AIP1sgRNAsp2 was the maximum. LentiCRISPRv2-sgRNAsp2 expression vector ofAIP1-knockout was successfully constructed, and infected 293T cells. The stable 293T cell lines withAIP1-expression-deficient were obtained by Western blot. In conclusion, the stable 293T cell lines ofAIP1-knockout were successfully generated by CRISPR/Cas9 system, which provides the foundation for further studying the functions ofAIP1gene.关键词
CRISPR/Cas9/慢病毒/AIP1/稳定细胞株Key words
CRISPR/Cas9/lentiviral/AIP1/stable cell lines引用本文复制引用
苏甲林,阙彪,张继勤,李锦辉,王敏,纪卫东..建立CRISPR/Cas9慢病毒系统高效敲除人源AIP1基因[J].生物技术通报,2015,(8):219-224,6.基金项目
国家自然科学基金项目 （）
