Smad3基因小干扰RNA的序列设计及其表达载体的构建

Designment and vector construction of siRNA sequences to Smad3 gene in vitro

目的构建大鼠Smad3基因小干扰RNA的表达质粒,为肝纤维化RNAi介导的基因治疗打下基础。方法利用互联网资源针对Smad3基因设计并合成三条可能的小干扰RNA,脂质体瞬时转染大鼠肝星状细胞,RT-PCR检测Smad3基因的表达情况。然后将这三条小干扰RNA插入到RNA干扰载体pRNAT-U6.1/Neo中,构建成三条干扰载体并测序。结果 RT-PCR和测序结果表明,成功构建了Smad3基因的RNA干扰真核表达载体。结论重组质粒pRNAT-SiSmad3的成功构建,为后续的Smad3基因下调实验提供了可能。

Objective To construct an expression vector with small interfering RNAs of Smad3 in vitro.Methods Three possible small interfering RNAs to Smad3 gene were designed and synthesized,subsequently transfected into rat hepatic stellate cells using Liposome 2000.RT-PCR was used to detect the expression of the Smad3 gene.Then the strands were cloned into pRNAT-U6.1/Neo vector to construct the recombinant plasmids and confirmed by sequencing.Results Both RT-PCR and sequencing assays showed that the recombinant vector pRNAT-SiSmad3 was successfully constructed.Conclusion The successful construction of the recombinant plasmid pRNAT-SiSmad3 will be beneficial to further study in the experiment of Smad3 down-regulation.