Detection of transfection efficiency of TIMP-1 targeted siRNA eukaryotic expression vectors in hepatic stellate cells

Objective To improve the transfection efficiency of pGCsi-U6/TIMP-1 shRNA eukaryotic expression vectors in HSC-T6 cell line.Methods Plasmids pGCsi-U6/TIMP-1 shRNA containing green fluorescent protein(GFP) gene was constructed,and then the plasmids pGCsi-U6/TIMP-1 shRNA transfected HSC-T6 cell line.According to the different ratio of plasmid DNA/Lipofectamine 2000(Life technologies),the transfection protocols were divided into 5 groups: naked DNA,2 μg/4 μL,2 μg/6 μL,2 μg/8 μL,2 μg/10 μL.Forty-eighty hours after transfection,transfection efficiencies of different protocols were evaluated by Laser Scanning Confocal Microscope and flow cytometer counting.Results The best transfection efficiency could be got when the ratio of plasmid DNA/Lipofectamine 2000 was 2 μg/8 μL compared with other groups.There was a significant difference,which showed that transfection efficiency was correlated with the ratio of plasmid to lipofectamine.Conclusion The efficiency of transferring TIMP-1 pGCsi-U6/shRNA into HSC-T6 can be higher with proper ratio of plasmid to lipofactamine.TIMP-1 pGCsi-U6/shRNA is an ideal transient expression vector for HSC-T6 gene transference.