RNA靶向的CRISPR/CasRx系统在小鼠精原细胞系GC1-spg中的应用  

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

作　　者：李梦真 柳俊 邹定峰 缪时英[1] 王琳芳[1] 宋伟[1] 李凯 LI Meng-zhen;LIU Jun;ZOU Ding-feng;MIAO Shi-ying;WANG Lin-fang;SONG Wei;LI Kai(State Key Laboratory of Medical Molecular Biology,Institute of Basic Medical Sciences CAMS,School of Basic Medicine PUMC,Beijing 100005,China)

机构地区：[1]中国医学科学院基础医学研究所,北京协和医学院基础学院,医学分子生物学国家重点实验室,北京100005

出　　处：《基础医学与临床》2021年第5期653-660,共8页Basic and Clinical Medicine

基　　金：国家自然科学基金(31970794,32000586)。

摘　　要：目的探究CRISPR/CasRx介导的RNA水平的基因敲降在小鼠精原细胞系GC1-spg的应用。方法利用同源重组的方法构建EF1a core promoter.CasRx.SV40.U6.DRs表达质粒(CasRx-gRNA)和EF1a.EGFP、EF1a.mCherry、EF1a.tdToamto 3种荧光蛋白表达质粒。在人胚肾细胞系HEK-293T内瞬时转染3种荧光蛋白表达质粒和CasRx-gRNA表达质粒,通过荧光强度、Western blot检测外源基因(荧光蛋白、EGFP和mCherry)的敲降情况。在HEK-293T细胞内转染CasRx-gRNA,通过q-PCR检测内源基因mRNA和lncRNA干扰后的表达水平。在小鼠精原细胞系GC1内瞬时转染外源基因egfp、mCherry和CasRx-gRNA表达质粒并通过以上方法检测外源基因(荧光蛋白)沉默效率。结果成功构建了CasRx-gRNA和3种荧光蛋白表达质粒。在HEK-293T细胞内CRISPR/CasRx介导的RNA干扰外源基因(egfp、mCherry)和内源基因(mRNA、lncRNA)后,表达水平均显著下调(P<0.01)。在小鼠精原细胞系GC1内验证CRISPR/CasRx系统,可有效和特异敲降外源基因egfp、mCherry。结论CRISPR/CasRx系统能够在GC1-spg细胞中发挥有效和特异的敲降作用,为雄性生殖发育的研究提供新的基因沉默工具。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 detected by the above method.Results CasRx-gRNA and three kinds of fluorescent protein expression plasmids were constructed successfully.After CRISPR/CasRxmediated RNA interference of exogenous genes(egfp,mCherry)and endogenous genes(mRNA,lncRNA)in HEK-293T cells,the expression level was significantly reduced(P<0.01).The CRISPR/CasRx system was verified in the mouse spermatogonia cell line GC1,which could effectively and specifically knock down exogenous genes egfp and mCherry.Conclusions CRISPR/CasRx system exerts an effective and specific knock-down function in GC1-spg,providing a new gene silencing tool potentially used in the study of male reproduction.

关 键 词：CRISPR/CasRx RNA 敲降 GC1-spg 

分 类 号：R34[医药卫生—基础医学]