肿瘤转移抑制蛋白MTss1 shRNA慢病毒载体的构建与鉴定

Construction and Identification of Lentiviral RNA Interference Vector of Mouse MTssl Gene

肿瘤转移抑制蛋白MTss1在小脑浦肯野神经元发育中表达受到调控,然而功能并不清楚。拟通过RNAi的方法降低内源MTss1的表达,为MTss1在神经元中的功能研究建立基础。针对小鼠MTss1基因(Mtss1)的mRNA序列,设计合成三对shRNA序列,克降到pSuper-Retro-Puro质粒中,瞬间转染GFP-Mtss1持续表达的N1H 313细胞株,western blot结果表明三对shRNA序列均可有效抑制MTss1的表达。进一步从pSuper质粒上酶切含shRNA的片段,克隆到慢病毒载体pLVTHM中并与包装质粒和衣壳质粒共转293T细胞,收集含病毒的上清液,测定感染效率。结果表明成功构建抑制MTss1基因表达的shRNA慢病毒载体,为内源MTss1的功能研究提供基础。

Metastasis suppressor 1 MTss1 , as a developmentally regulated gene, has been implicated in the neurogenesis of cerebellar purkinje neuron but the function remains unclear. The aim in this study is to knock down the expression of endogenous MTss1 through RNA interference strategy. Three pair of target sequence were designed and synthesized, then cloned into pSuper-Retro-Puro vector. The knock down efficiencies were further checked by transient transfection and western blot. The shRNA containing fragments from pSuper-Retro-Puro plasmids were released and further cloned into lentiviral vector. The infection efficiencies of RNAi lentivirus were determined by western blot and fluorescence microscopy. The successful construction of mouse lentiviral vectors expressing mouse MTss1-specific shRNA should be use- ful for further in vivo study.