CRISPR/Cas9慢病毒系统敲除胰岛β细胞PKA C-α的研究

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

摘要/Abstract

摘要： 利用CRISPR/cas9系统建立稳定敲除PKA C-α基因的INS-1细胞株,研究PKA C-α在胰岛β细胞中的功能。设计2个长25 bp且分别靶向PKA C-α基因的exon 5和exon 7的sgRNA,将其克隆至LentiCRISPRv2-sgRNA质粒并转染至293T细胞中制备sgRNA-Cas9慢病毒,慢病毒感染INS-1细胞,嘌呤霉素筛选出阳性细胞并采用有限稀释法筛选单克隆细胞,Western blotting印记法检测单克隆细胞中PKA C-α蛋白的表达水平,测序确认单克隆细胞中PKA C-α基因的突变位点。WB实验证实靶向Exon 5的sgRNA可成功敲除PKA C-α基因,得到稳定敲除PKA C-α基因的细胞株,测序结果表明该细胞株的PKA C-α基因发生1 bp碱基插入突变,并且敲除PKA C-α基因的INS-1细胞胰岛素分泌能力下降。本实验利用CRISPR/Cas9系统成功敲除INS-1细胞中的PKA C-α基因,为研究PKA C-α在胰岛β细胞中的功能奠定了基础。

关键词: CRISPR/cas9系统, PKA C-α, , INS-1细胞, 稳定细胞株

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

何士俊, 万毅虹, 章嘉雯, 蔡秀潮, 刘静文, 刘叔文, 姚新刚. CRISPR/Cas9慢病毒系统敲除胰岛β细胞PKA C-α的研究[J]. 生物技术通报, 2020, 36(3): 102-109.

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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