摘要
目的构建慢病毒表达小鼠CARM1基因shRNA的质粒,观察其在小鼠畸胎瘤P19细胞中对CARM1的表达抑制效率。验证CARM1 shRNA慢病毒表达质粒,进一步筛选得到稳定敲低CARM1的小鼠畸胎瘤P19细胞系。方法设计并且合成针对小鼠CARM1 mRNA不同部位的4组shRNA序列,以AgeⅠ和EcoRⅠ克隆入pLKO.1慢病毒载体中,得到含shRNA的慢病毒表达质粒。氨苄青霉素筛选后,DNA测序鉴定结果。鉴定正确的质粒大量制备后,与慢病毒包装辅助质粒psPAX2、pMD2.G共同转染HEK293T细胞,包装得到具有感染能力的慢病毒。将含病毒的培养基上清感染P19细胞后,经嘌呤霉素筛选获得CARM1稳定敲低的P19细胞系,并用Western blot法和实时荧光定量PCR进行鉴定。结果测序鉴定表明4组CARM1 shRNA慢病毒表达质粒构建成功。Western blot法检测和实时荧光定量PCR分析结果显示4组CARM1 shRNA慢病毒表达载体中的2组可有效地抑制小鼠畸胎瘤P19细胞中CARM1基因的表达,即pLKO.1/CARM1 shRNA2、pLKO.1/CARM1 shRNA3有明显的抑制效果。结论成功构建了针对CARM1基因的shRNA的慢病毒表达质粒并筛选得到稳定敲低CARM1表达的P19细胞系,为进一步研究CARM1基因的功能奠定了基础。
Objective To design the small hairpin RNA （shRNA） targeting CARM1 and inserte into the lentiviral expression vectors and to identify their inhibition efficacy on CARM1 in teratocarcinoma P19 Cells.To establish P19 cell line stable knockdown of CARM1 gene by transduced the cells with constructed viral products.Methods Four different shRNA sequences targeting CARM1 gene were designed.The corresponding DNA fragments were synthesized in vitro,annealed and then cloned into the pLKO.1 vector.The lentiviral plasmids were confirmed by DNA sequencing,then transfected into HEK293T cells to package into lentiviral particles.The harvested supernatant with lentiviral particles was transduced into P19 cells and selected by puromycine.Western blot and Quantitative real-time PCR analysis were carried out to analyze the suppression of CARM1 in P19 Cells.Results DNA sequencing showed CARM1 shRNA lentiviral plasmids were successfully constructed.Western blot and Quantitative real-time PCR analysis confirmed the effective knockdown of CARM1 by two shRNA.Conclusion A stable P19 cell line has been successively established,in which CARM1 expression is significantly inhibited.
出处
《医学研究杂志》
2014年第7期38-43,共6页
Journal of Medical Research
基金
教育部博士点(博导类)基金资助项目(20111106110024)
