Methods:Design and synthesize three siRNAs based on the sequence of ER-âmRNA. They were separately subcloned into the plasmid of pSliencer 3. 1-H1 containing U6 promoter. The pSliencer 3. 1-H1 vectors of the RNA interference eukaryotic expression specific to ER-âgene were constructed. The pSliencer 3. 1-H1 vectors were transfected into bone mesenchyma cells with Lipofectine 2000 in vitro. The positive cell clones were obtained after being screened with 200ìg/ml G418. Then the effects of RNAi on the expression of ER-â gene were detected by RT-PCR and Western Blot . The cellular growth activities were assayed by flow cytometry in vitro.
Results:DNA sequencing of the plasmids verified the successful construction of the ER-â siRNA vectors. Compared with the blank and the control group , the expressions of ER-â mRNA and protein were remarkably down regulated in the RNAi groups and the cell proliferations had no significance change in the RNAi groups.
Conclusion:We successfully construct the recombinant plasmids of siRNAs specific to ER-âgene and transfect them into the bone mesenchyma cells in vitro. The RNAi inhibits the expression of ER-â mRNA and protein , and no suppresses the proliferation of bone mesenchyma cells.