Lentiviral CRISPR/Cas9-Mediated PKA C-α Knockout in Pancreatic-β Cell

doi:10.13560/j.cnki.biotech.bull.1985.2019-0534

Abstract

Abstract: This work aims to establish the INS-1 cell line with PKA C-α stably knocked out using CRISPR/ Cas9 technique and to study the function of PKA C-α in pancreatic β-cell. Two 25 bp sgRNAs designed to target exon 5 and exon 7 of PKA C-α separately were chemically synthesized and inserted into LentiCRISPRv2 plasmid.,the plasmid was then transfected to 293T cells to prepare sgRNA-Cas9 lentivirus,and the lentivirus was then infected INS-1 cells. The positive cells were screened using puromycin and the monoclonal cells were obtained using infinite dilution method. The expression of PKA C-α protein was detected by Western blotting assay in monoclonal cells and the mutation site was confirmed by sequence analysis. It was confirmed by Western blotting assay that the exon 5-trageting sgRNA successfully knocked out PKA C-α.The INS-1 cell line with PKA C-α gene stably knockout was obtained. Sequence analysis results showed a 1-bp insertion mutation in the PKA C-α gene occurred and insulin secretion of INS-1 with PKA C-α gene knockout reduced. In sum,knocking out the gene PKA C-α using the CRISPR/Cas9 system in INS-1 cell line is successful,which lays a foundation in studying the function of PKA C-α in pancreatic β-cell.

Key words: CRISPR/Cas9 system, PKA C-α, INS-1 cell, stable cell line

HE Shi-jun, WAN Yi-hong, ZHANG Jia-wen, CAI Xiu-chao, LIU Jing-wen, LIU Shu-wen, YAO Xin-gang. Lentiviral CRISPR/Cas9-Mediated PKA C-α Knockout in Pancreatic-β Cell[J]. Biotechnology Bulletin, 2020, 36(3): 102-109.

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