Application of RNA-targeted CRISPR/CasRx system in mouse spermatogonia cell line GC1-spg

• Published in: Ji chu yi xue yu lin chuang = Jichu yixue yu linchuang = Basic medical sciences and clinics Vol. 41; no. 5; pp. 653 - 660
• Main Author: LI Meng-zhen, LIU Jun, ZOU Ding-feng, MIAO Shi-ying, WANG Lin-fang, SONG Wei, LI Kai
• Format: Journal Article
• Language: Chinese
• Published: Institute of Basic Medical Sciences and Peking Union Medical College Hospital, Chinese Academy of Medical Sciences / Peking Union Medical College 01.05.2021
• Subjects: crispr/casrx|rna|knockdown|gc1-spg
• ISSN: 1001-6325

Objective To investigate the application of CRISPR/CasRx mediated gene knockdown at the RNA level in mouse spermatogonia cell line GC1-spg. Methods The EF1a core promoter. CasRx.SV40.U6.DRs expression plasmid (CasRx-gRNA) and three fluorescent protein of EF1a.EGFP, EF1a.mCherry, EF1a.tdToamto expression plasmids were constructed by homologous recombination. Three fluorescent protein expression plasmids and CasRx-gRNA expression plasmids were transiently transfected into human embryonic kidney cell line HEK-293T, and the knockdown of exogenous gene (the fluorescent proteins,EGFP and mCherry) was used to detect by fluorescence intensity and Western blot. Additionally, the expression levels of endogenous gene (mRNA and lncRNA) were measured by q-PCR in HEK-293T cells transfected with CasRx-gRNA. The exogenous gene egfp, mCherry and CasRx-gRNA expression plasmids were transiently transfected into the mouse spermatogonia cell line GC1, and the knockdown efficiency of exogenous gene (the fluorescent proteins) was d