Establishment of a CRISPR/Cas9 Lentiviral System for Knockout Gene AIP1 of Human

doi:10.13560/j.cnki.biotech.bull.1985.2015.08.032

Abstract

Abstract: This work aims to establish the CRISPR/Cas9 system of knocking out gene AIP1 for producing nephocyte cell（293T）of human embryo in which gene AIP1 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 genes AIP1 were knocked out successfully. The expression level of AIP1 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 of AIP1-knockout was successfully constructed, and infected 293T cells. The stable 293T cell lines with AIP1-expression-deficient were obtained by Western blot. In conclusion, the stable 293T cell lines of AIP1-knockout were successfully generated by CRISPR/Cas9 system, which provides the foundation for further studying the functions of AIP1 gene.

Key words: CRISPR/Cas9, lentiviral, AIP1, stable cell lines

Su Jialin, Que Biao, Zhang Jiqin, Li Jinhui, Wang Min, Ji Weidong. Establishment of a CRISPR/Cas9 Lentiviral System for Knockout Gene AIP1 of Human[J]. Biotechnology Bulletin, 2015, 31(8): 219-224.

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