小鼠Retn基因siRNAs的设计及其稳定表达载体的构建

目的:设计小鼠Retn基因特异性的siRNAs,并构建一系列能在哺乳动物细胞内稳定表达这些siRNAs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pSilencerEM1.0-Neo载体;(2)用电穿孔转染和G418筛选等方法建立能稳定表达相应siRNAs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mRNA水平.结果:设计并构建了4个小鼠Retn基因特异性的siRNAs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siRNAs,显著地抑制了这些脂肪细胞内Retn基因的mRNA水平.结论:本研究设计并构建出的小鼠Retn基因特异性的siRNAs表达载体,所表达的siRNAs具有较强的RNA干涉功能,为Retn基因的功能研究奠定了实验基础.

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

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.