小鼠Retn基因siRNAs的设计及其稳定表达载体的构建  

Design of mouse Retn-specific siRNAs and development of a vector-based siRNAs expression system

作　　者：李雅慧[1] 黎怀星[2] 吴丹[2] 施柯[2] 蔡东联[1] 孙树汉[2] 

机构地区：[1]长海医院营养科,上海200433 [2]第二军医大学基础医学部医学遗传学教研室,上海200433

出　　处：《第二军医大学学报》2004年第2期136-139,共4页Academic Journal of Second Military Medical University

基　　金：国家自然科学基金 ( 3 0 3 70 80 2 )

摘　　要：目的 :设计小鼠 Retn基因特异性的 si RNAs,并构建一系列能在哺乳动物细胞内稳定表达这些 si RNAs的表达质粒 ,以便为在体外研究 Retn基因的功能打下基础。 方法 :(1 )设计并合成小鼠 Retn基因特异性的一组寡核苷酸片段 ,并克隆到 p Silencer EM 1 .0 - N eo载体 ;(2 )用电穿孔转染和 G4 1 8筛选等方法建立能稳定表达相应 si RNAs的一组 3T3- L1细胞克隆 ;(3)诱导细胞分化为脂肪细胞 ,并用 RT- PCR分析这些脂肪细胞内 Retn基因的 m RNA水平。 结果 :设计并构建了 4个小鼠Retn基因特异性的 si RNAs表达质粒 ,并证明其中的 2种质粒能在脂肪细胞内稳定表达相应的 si RNAs,显著地抑制了这些脂肪细胞内 Retn基因的 m RNA水平。 结论 :本研究设计并构建出的小鼠 Retn基因特异性的 si RNAs表达载体 ,所表达的 si R-NAs具有较强的 RNA干涉功能 ,为Objective:To design mouse Retn specific siRNAs and construct a group of plasmids for expressing various mouse Retn specific siRNAs in mammalian cells. Methods: A group of ds oligonucleotides fragments were synthesized and cloned into pSilencer EM 1.0 Neo vector. Electroporation and G418 screening were used to establish 3T3 L1 cell lines for stable expression of mouse Retn specific siRNAs. The cell line was induced to differentiate into adipocyte and mRNA levels of Retn gene in the adipocytes were analyzed using RT PCR. Results: Four plasmids used for expressing mouse Retn specific siRNAs have been designed and construced, and 2 of them were proven to be able to express siRNAs and to inhibit mRNA level of mouse Retn gene in adipocytes. Conclusion: The vectors constructed are able to express the mouse Retn specific siRNAs and they are potent in mediating Retn specific RNA interference in adipocytes.

关 键 词：小鼠 Retn基因 SIRNAS 设计 载体 基因表达 脂肪细胞 

分 类 号：R394[医药卫生—医学遗传学] R-33[医药卫生—基础医学]