内源性MGF在IGF-I Pre-mRNA选择性剪接中的作用  

Effects of Endogenous Mechano-growth Factors on Alternative Splicing of IGF-I pre-mRNA

作　　者：梁培龙 李金桥[1] 杨超[1] 陈永杰[1] 吴峰[1] 张洪玉[1] 李莹辉[1] 戴钟铨[1] 万玉民[1] 

机构地区：[1]中国航天员科研训练中心,航天医学基础与应用国家重点实验室,北京100094

出　　处：《航天医学与医学工程》2016年第2期79-83,共5页Space Medicine & Medical Engineering

基　　金：国家自然科学基金项目(31470832);国家重大科学仪器设备开发专项(2013YQ190467);航天医学基础与应用国家重点实验室项目(SMFA12B02);中国航天医学工程预先研究项目(2012SY54A1602)

摘　　要：目的利用CRISPR/Cas9敲除小鼠成肌细胞系C2C12机械生长因子(mechano-growth factors,MGF),研究MGF在IGF-I pre-mRNA选择性剪接和C2C12增殖分化中的作用。方法针对IGF-I第五个外显子设计并构建CRISPR/Cas9基因敲除质粒,通过细胞转染并筛选出MGF发生移码突变的细胞株。采用RT-q PCR技术,检测这些细胞株MGF和IGF-IEa表达变化,以及细胞增殖和肌向诱导分化能力的变化。结果成功构建了针对MGF的CRISPR/Cas9基因敲除质粒,并筛选出3株MGF移码突变细胞株。这些细胞株的MGF和IGF-IEa表达上升,细胞增殖活性减弱,在诱导分化过程中,肌细胞标志性基因myogenin、MGF和IGF-IEa表达升高。结论内源性MGF敲除能够影响IGF-I pre-mRNA的选择性剪接以及增殖分化能力。Objective To explore the effects of endogenous MGF knockout on IGF-I pre-mRNA alternative splicing,the proliferation and differentiation of C2C12. Methods MGF was knockout in mice myoblast cell line C2C12 with CRISPR / Cas9. A gRNA targeted the fifth exon of IGF-I was designed and constructed into CRISPR / Cas9 vector. Monoclonal cell lines with MGF frameshift mutation were screened via cell transfection.MGF and IGF-IEa expression were measured with RT-q PCR. The proliferation and differentiation changes were detected in these cells. Results MGF knockout plasmid was successfully constructed with CRISPR / Cas9,and three monoclonal cell lines with MGF frameshift mutation were screened out. In these cells,MGF and IGF-IEa expression were increased,proliferation activity were decreased,and in the process of differentiation MGF,IGF-IEa and muscle cell＇s marker myogenin expression were increased. Conclusion Endogenous MGF knockout can influence IGF-I pre-mRNA alternative splicing and the proliferation and differentiation of C2C12.

关 键 词：CRISPR/Cas9 机械生长因子MGF IGF-I 选择性剪接 

分 类 号：Q789[生物学—分子生物学]