Noggin基因沉默表达载体的构建及效果评价  

Construction and evaluation of gene silencing vectors of Noggin

作　　者：马雨楠 游颖[1] 孙兆增[1] 曾林[1] 

机构地区：[1]军事医学科学院实验动物中心,北京100071

出　　处：《中国实验动物学报》2016年第1期37-42,共6页Acta Laboratorium Animalis Scientia Sinica

基　　金：国家自然科学基金重点项目(31030058);国家科技支撑计划(2011BA115B03)

摘　　要：目的构建慢病毒介导的Noggin RNAi干扰序列,并分析这些干扰序列对Noggin基因的沉默效果。方法针对目的基因Noggin的m RNA设计四条干扰序列,并将这些序列连接到Lenti-KD慢病毒载体,将重组质粒瞬时转染HEK-293T包装细胞,获得重组慢病毒。将重组病毒感染MC3T3-E1细胞,利用puromycin进行筛选,获得稳定表达细胞系。通过实时荧光定量PCR和Western blot技术分析不同干扰序列的干扰效果。结果实时荧光定量PCR结果显示,四种干扰序列对Noggin基因的表达都有一定的沉默效果,但只有sh Noggin-1(P<0.01)对其表达影响显著。Western blot结果显示,四种干扰序列中只有sh Noggin-1(P<0.01)对Noggin的表达蛋白具有显著的降低作用。结论获得了一种Noggin基因的干扰序列,该序列能够干扰Noggin基因m RNA的稳定性,从而影响蛋白的表达。该干扰序列可以用于部分敲除Noggin基因,从而用于研究Noggin基因的功能。Objective To construct the retroviral-mediated short hairpin RNA（ sh RNA） expression vectors of Noggin,and to analyze the silencing effect of the sh RNA. Methods Four interference sequences were designed based on Noggin m RNA,and connected to Lenti-KD vectors. The recombinant plasmids were transiently transfected into HEK-293 T cells,and finally obtained the recombinant virus. Then MC3T3-E1 cells were infected with the recombinant virus and screened by puromycin. The gene silencing effect of sh RNA was evaluated using RT-PCR and Western blot. Results RTPCR results showed that all the four interference sequences had silencing effect on the expression of Noggin,but only sh Noggin-1（ P〈0. 01） was different significantly. Western blot results showed that among the four interference sequences,only sh Noggin-1（ P〈0. 01） showed a significantly lowering effect on the expression of Noggin. Conclusions One interference sequence of Noggin gene is obtained,which can interfere the stability of m RNA of the Noggin gene,by affecting the protein expression. It is useful for the further study of unknown biological function of Noggin.

关 键 词：Noggin基因 基因沉默 表达载体 Uncv无毛小鼠 

分 类 号：Q95-33[生物学—动物学]