Abstract: Objective To construct the eukaryotic expression plasmids of short hairpin RNA （shRNA） specific for mouse Bcl-2-associated athanogene 1 （BAG-1） and to observe their inhibitory effects on the expression of BAG-1 gene in mouse melanoma B16F10 cells. Methods Plasmids named pRNAT-U6.1/Neo-BAG-1, were designed and constructed to target the mouse BAG-1 mRNA coding region. LipofectaminTM 2000 was used to transfect plasmids into B16F10 cells. Negative plasmid-transfected and untransfected B16F10 cells served as negative and blank controls respectively. Forty-eight hours following transfection, G418 was used to select the resistant cells. The mRNA and protein expression of BAG-1 gene was measured by reverse transcription-PCR and Western blot respectively about 1 month after the transfection. Results The eukaryotic expression plasmids, pRNAT-U6.1/Neo-BAG-1, were constructed, and verified by restriction enzyme digestion and DNA sequencing. The transfection rate in B16F10 cells was 20%-30%. Compared with the blank control, the mRNA and protein expression of BAG-1 in B16F10 cells was significantly inhibited by BAG-1 shRNA （both P < 0.05）, and the inhibition rates were （77 ± 4）% and （62 ± 2）%, respectively. Conclusions These results indicate that the eukaryotic expression vectors containing shRNA against BAG-1 gene, pRNAT-U6.1/Neo-BAG-1, are successfully constructed, and can significantly inhibit the expression of BAG-1 gene in mouse melanoma B16F10 cells.

Key words: small hairpin RNA, BAG-1, melanoma