摘要
为构建Sirtl基因低表达的LMH细胞系,根据鸡Sirtl基因的3′UTR序列设计gSmiR30、gSmiR70和gSmiR913个miRNAs,以质粒pLNCX-gSmiRn(n为30,70或91)分别过表达这3个miRNAs48h后,依次检测其对CHO-K1细胞中报告载体psiCHECH2-gSirtl-UTR672所表达的荧光素酶活性的影响以及对LMH细胞中Sirtl基因表达的干扰情况;然后选用干扰效果较好的pLNCX-gSmiR30包装的反转录病毒感染LMH细胞,以终浓度1μg·mL^-1的G418筛选稳定过表达gSmiR30的LMH-gSmiR30细胞系,荧光定量PCR和Westernblot检测Sirtl基因的表达。结果表明:人工设计的3个miRNAs可抑制含靶位点的海肾荧光素酶活性在97%以上;且LMH细胞中Sirtl的mRNA表达水平均能被这3个miRNAs下调40%以上;筛选得到的LMH-gSmiR30细胞中绿色荧光蛋白的表达率在99%以上,细胞内Sirt1基因的mRNA水平下降75%,SIRT1蛋白也明显减少。这一研究成功获得了Sirt1基因低表达的LMH-gSmiR30细胞系,为进一步研究Sirt1基因在鸡肝脏中的功能提供了一种很好的细胞模型。
To generate the LMH cell line with low Sirtl expression, three miRNAs (gSmiR30, gSmiR70 and gSmiR91) were designed targeting to the 3′UTR sequence of chicken Sirtl gene. The luciferase activity was assessed after pLNCX-gSmiRn (n :30, 70 or 91) plasmids expressing the three miRNAs were co-transfected with the reporter vector psiCHECH2-gSirt1-UTR672 into CHO-Kl for 48 hours. Sirt1 gene expression was also detected to screen for the best effective sequence in LMH cells. Then, LMH cells were infected with the retroviral particles packaged with pLNCX- gSmiR30 with the best interference effect. LMH-gSmiR30 cells stably overexpressing gSmiR30 were screened with 1μg·mL^-1 G418. The expression of Sirtl gene was detected by RT-PCR and Western blot. The results showed that the three miRNAs could inhibit the activity of luciferase containing target sites by more than 97%. S/ril mRNA was also inhibited about 40% after three miRNAs were separately transfected in to LMH cells for 48 h. The expression ratio of the green fluorescent protein (GFP) was more than 99% in LMH-gSmiR30 after G418 resistance screening. Importantly, Sirtl expression was significantly knocked down in LMH-gSmiR30 cell lines, as indicated by the markedly decreased levels of Sirtl mRNA about 75% and protein expression. These results indicated that the LMH cells with low Sirtl expression were successfully obtained, which provided a good cellular model for further study of the the Sirtl gene function in chicken liver.
作者
郁建锋
胡悦
付修虎
王中亮
徐璐
龚道清
顾志良
YU Jianfeng;HU Yue;FU Xiuhu;WANG Zhongliang;XU Lu;GONG Daoqing;GU Zhiliang(College of Animal Science and Technology,Yangzhou University ,Yangzhou 225009, China;School of Biology and Food Engineering, Changshu Institute of Technology, Changshu 215500, China)
出处
《扬州大学学报（农业与生命科学版）》
CAS
北大核心
2019年第2期76-82,共7页
Journal of Yangzhou University：Agricultural and Life Science Edition
基金
国家自然科学基金资助项目(31472091、31772593)
