Construction and identification of the recombinant adenovirus expressing BMP4-siRNA

Objective To construct a recombinant adenovirus expressing siRNA target sites for human BMP4.Methods Six pairs of oligonucleotides containing siRNA target sites for human BMP4 using Dharmacon's siDESIGN program were subcloned into the Sfi Ⅰ site of pSOS,which has been inserted the coding regions of human BMP4,resulting in pSOS-sirBMP4.Then the pSOS-sirBMP4 were transfected into HEK-293 cells,and GFP signal level was used to assess the silencing efficiency of different siRNA target sites.Four pairs of oligonucleotides were chosed and subcloned into shuttle plasmid pSES-HUS and authenticity of PCR amplified sequences and the oligonucleotide cassettes were verified by DNA sequencing.The correctly identified recombinant plasmid was linearized with Pme Ⅰ and transformed into BJ5183-Ad-easy competent cells containing adenovirus backbone vector to produce recombinant adenovirus DNA by homologous recombination.Then the recombinant adenovirus DNA was linearized with Pac Ⅰ and transfected into 293 cells to make adenovirus.the silencing efficiency of recombinant adenovirus was assess by RT-PCR and Western blot in MDA-MB-231 cell line.MTT assay was used to detect the proliferation of MDA-MB-231 cells transfected with pAd-sirBMP4.Results Four pairs of oligonucleotides with high silencing efficiency were chosed and subcloned into shuttle plasmid pSES-HUS to make recombinant adenovirus.After infected with the recombinant adenovirus BMP4-siRNA,the expression of BMP4 sharply decrease in the human breast cancer cell line MDA-MB-231.Conclusions The recombinant adenovirus expressing siRNA target sites for human BMP4 has been successfully constructed and may be used to silence the expression of BMP4 in MDA-MB-231 cell line effectively.