抑制骨髓基质干细胞雌激素受体β基因表达的有效序列

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BamH Ⅰ(GATCC)和Hind Ⅲ (AGCTT)酶的酶切位点，最后形成两条互补的克隆入pSilencer 3.1-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。

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

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.