抑制骨髓基质干细胞雌激素受体β基因表达的有效序列

Construction and identification of eukaryotic expression vector of RNA interference specific for estrogen receptor beta

背景:雌激素受体β是否参与介导骨髓间充质干细胞的增殖与分化需进一步实验论证。目的:以RNAi技术寻找和验证对大鼠骨髓基质干细胞雌激素受体β基因表达抑制的有效序列。方法:根据GeneBank数据库提供的SD大鼠雌激素受体β基因核苷酸序列,选择设计能转录小发卡结构RNA(Small hairpin RNAs,shRNA)的DNA序列。再在两条互补碱基序列的5'端分别加上BamHⅠ(GATCC)和HindⅢ(AGCTT)酶的酶切位点,最后形成两条互补的克隆入pSilencer3.1-H1载体的发夹状siRNA模板序列,进行重组载体的碱基序列测定。结果与结论:重组质粒碱基序列鉴定后,证实真核表达载构建正确。雌激素受体β特异性siRNA真核表达载体构建成功。

BACKGROUND： Whether estrogen receptor beta （ER-β） participates to mediate proliferation and differentiation of bone marrow mesenchymal stem cells is unclear.OBJECTIVE： To construct and identify a specific small interfering RNA （siRNA） suppressing estrogen receptor beta gene.METHODS： Genome sequences of ER-β gene was retrieved from Genbank and cDNA was designed coding expression of small hairpin RNAs （shRNA） for ER-β gene.It was cloned into the expression vector pSilencer 3.1-H1 into a short hairpin RNA with BamH Ⅰ,HindⅢ sticky end,terminated cognition sequence and a loop structure,followed by being cloned in pRNAT-U6.1 /Neo to construct a expression vector of ER-β-specific siRNA.At last the vector was evaluated by enzyme cutting and gene sequencing test.RESULTS AND CONCLUSION： The recombinant plasmid of RNA interference specific for ER-β identified by the restriction map completely coincided with the designs.The siRNA eukaryotic expression vector against ER-β mRNA has been successfully constructed.