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。
This work aims to establish the INS-1 cell line with PKA C-αstably knocked out using CRISPR/Cas9 technique and to study the function of PKA C-αin pancreaticβ-cell.Two 25 bp sgRNAs designed to target exon 5 and exon 7 of PKA C-αseparately 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-αprotein 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-αgene stably knockout was obtained.Sequence analysis results showed a 1-bp insertion mutation in the PKA C-αgene occurred and insulin secretion of INS-1 with PKA C-αgene knockout reduced.In sum,knocking out the gene PKA C-αusing the CRISPR/Cas9 system in INS-1 cell line is successful,which lays a foundation in studying the function of PKA C-αin pancreaticβ-cell.
作者
何士俊
万毅虹
章嘉雯
蔡秀潮
刘静文
刘叔文
姚新刚
HE Shi-jun;WAN Yi-hong;ZHANG Jia-wen;CAI Xiu-chao;LIU Jing-wen;LIU Shu-wen;YAO Xin-gang(Guangdong Provincial Key Laboratory of Drug Screening,Guangzhou Key Laboratory of Emerging Virus,School of Pharmacy,Southern Medical University,Guangzhou 510515)
出处
《生物技术通报》
CAS
CSCD
北大核心
2020年第3期102-109,共8页
Biotechnology Bulletin
基金
国家自然科学基金项目(81603118)
广州市“珠江新星”(201806010119)
广东省大学生创新创业项目“攀登计划”(pdjh2017b0098)
