雌激素受体α亚基基因小干扰RNA表达载体的构建

Construction of specific siRNA expression vector of estrogen receptor α subunit gene

目的探讨构建雌激素受体α（ERα）亚基基因小干扰RNA（siRNA）表达载体的方法，为建立基因敲除ERα亚基突变株提供素材和依据。方法利用计算机辅助设计ERα特异性siRNA，体外合成siRNA基因，并将其定向克隆入pSilencer4．1-CMV质粒中，构建真核表达载体。采用PCR扩增、酶切分析鉴定。结果双酶切法鉴定重组质粒后，RT-PCR法鉴定ERasiRNA载体可特异地抑制人成骨样细胞株MG63细胞中ERα的表达。结论ERα亚基特异性siRNA表达载体被成功构建，为进一步研究雌激素受体在骨代谢中的作用机制提供了实验素材和依据。

Objeαive To explore the construαion of specific siRNA expression veαor of estrogen receptor α（ERα） subunit expression, and to provide materials and evidence for knocking out the gene of ERα subunit of mutant strain. Methods According to the computer aided design （CAD）, ERα specific siRNA gene was synthesized and cloned into the expression veαor pSilencer 4.1-CMV. The construαed ERα siRNA was transfeαed into human osteoblast-like cell line MG63 and the inhibition effeα of ERα siRNA on expression of ERα in MG63 cells was deteαed using the semiquantitive RT-PCR. Results Double enzyme digestion analysis confirmed that the ERα specific siRNA expression veαor was construαed successfully. ERα expression in MG63 cells was specifically suppressed after transfeαion of ERα siRNA. Conclusion The ERα specific siRNA expression veαor is construαed successfully, which may provide a novel applicable strategy for estrogen receptor in osteoporosis.