摘要
目的：构建针对RhoA基因的shRNA表达载体并进行鉴定。方法：针对人RhoA的mRNA序列，设计并合成编码的shRNA的两条寡核苷酸序列，经退火成互补双链，再克隆至pGPU6／GFP／Neo质粒，构建重组体pGPU6／GFP／Neo—RhoA，进行酶切及测序鉴定，然后脂质体转染LoVo细胞。荧光显微镜观察绿色荧光蛋白的表达。结果：将合成的DNA序列退火后克隆到载体，酶切及测序证实质粒为所需的序列。结论：成功地构建了针对RhoA基因的shRNA表达载体，为下一步进行RNAi的相关研究奠定了基础。
AIM： To construct and identify shRNA inter- ference vector targeting RhoA gene. METHODS ： Two pairs of single stranded oligonueleotides encoding RhoA siRNA sequence were designed and synthesized. After annealing, its were inserted into vector pGPU6/GFP/Neo, constructed recombinant vectors and then were identified by restrictive digestion and DNA sequencing. LoVo cells were transfected with the recombinant DNA samples by the liposome complex method, and then fluo-rescence photographs were taken. RESULTS： Enzyme digestion and DNA sequencing showed that the oligonucleotide fragmentswere correctly inserted into pGPU6/GFP/Neo vector. CONCLUSION： The RhoA genetargeted siRNA and its vector are successfully constructed.
出处
《细胞与分子免疫学杂志》
CAS
CSCD
北大核心
2010年第7期707-708,共2页
Chinese Journal of Cellular and Molecular Immunology
基金
湖南省科学技术厅科技计划项目(2008FJ3160)
