Silencing BAO-1 gene by eukaryotic expression vector containing short hairpin RNA in mouse melanoma B16F10 cells
- VernacularTitle:短发卡RNA对黑素瘤B16F10细胞BAG-1基因表达的抑制作用
- Author:
Yali SONG
;
Hao CHEN
;
Yi LIU
;
Jia CHEN
;
Yanning XUE
;
Xuesi ZENG
;
Jianfang SUN
- Publication Type:Journal Article
- Keywords:
Melanoma;
RNA,small interference;
Genes,BAG-1
- From:
Chinese Journal of Dermatology
2008;41(9):594-597
- CountryChina
- Language:Chinese
-
Abstract:
Objective To construct the eukaryotic expression plasmids of short hairpin RNA (shRNA) specific for mouse Bcl-2-assoeiated athanogene 1 (BAG-1) and to observe their inhibitory effects on the expression of BAG-1 gene in mouse melanoma B16FI0 ceils. 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 BI6F10 cells. Negative plasmid-transfected and tmtransfected 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 BI6FI0 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.
