CDC6靶向RNA干扰质粒载体的构建及生物活性鉴定  

Construction and Identification of CDC6 Targeting siRNA Expressing Plasmid

作　　者：还勇为[1] 陈平[1] 朱瑾[2] 闫洪涛[1] 何中林[1] 陈胤[1] 

机构地区：[1]第三军医大学大坪医院野战外科研究所肝胆外科,重庆400042 [2]第三军医大学西南医院全军肝胆外科研究所,重庆400038

出　　处：《现代生物医学进展》2007年第10期1500-1502,共3页Progress in Modern Biomedicine

摘　　要：目的:构建小鼠CDC6基因的RNAi真核表达载体PGCsilencer TM u6/Neo/GFP/RNAi,观察其转染小鼠肝细胞前后CDC6的表达变化。方法:根据GenBank中CDC6的序列,设计特异性siRNA序列,将模板序列克隆至PGCsilencer U6/Neo/GFP质粒中,通过测序鉴定后,用脂质体将重组子转染至正常小鼠肝细胞中,用RT-PCR检测CDC6的mRNA的表达及用Western blot方法检测CDC6蛋白水平的表达,并比较转染前后其表达水平的变化。结果:经测序,模板序列与设计序列完全正确,经过RT-PCR及Western blot方法检测,转染干扰质粒后,小鼠肝细胞中CDC6表达在mRNA及蛋白水平都有明显的下降。结论:成功构建了CDC6基因的RNAi真核表达载体并转染至小鼠肝细胞中,为下一步探讨CDC6在肝再生的作用奠定了基础。Objective： To build CDC6 targeting siRNA expressing plasmid, and explore the significance of CDC6 inhibition in hepatocytes, Methods： The specific siRNA sequence was designed according to the CDC6 sequence in GenBank. The sequence was cloned into PGCsilencerTM U6/Neo/GFP and then sequence analysis was performed. The recombinant plasmid was transfected into hepatocytes of mouse by Limpofectin2000. The mRNA expression of CDC6 gene was measured by RT-PCR,and the CDC6 protein was detected by Western blot method. Results： The plasmid PGCsilencerTMU6/Neo/GFP/RNAi could efficiently reduce the expression of CDC6 in mouse hepatocytes. Conclusions： The RNAi vector PGCsilencerTMU6/Neo/GFP/RNAi was constructed successfully. The RNAi inhibitory effect on the hepatocytes is confirmed. The successful construction of CDC6 RNAi makes it possible for the further studies of the effect of CDC6 on liver regeneration.

关 键 词：CDC6 RNA干扰 质粒 载体构建 

分 类 号：Q78[生物学—分子生物学] Q813