Abstract: Objective To compare the efficiency and cytotoxicity of different transfection reagents used in transfection of
RIP140-siRNA into Kupffer cells to optimize the transfection conditions. Methods Kupffer cells were transfected with
RIP140-siRNA labeled with GFP as the reporter gene using lipofectamine 2000, Roche reagent (X-treme GENE siRNA
Transfection Reagent) and puro screening lentivirus (1.0×108 TU/mL) as the transfection reagents. The transfection effect was
observed under a fluorescent inverted microscope, and laser scanning confocal microscopy was used to analyze RIP140
expression in trasnfected Kupffer cells. Flow cytometry was performed to detect cell apoptosis, and CCK-8 test was used to
evaluate the cell proliferation inhibition. RT-RCR and Western blotting were performed to detect the expressions of RIP140
mRNA and protein in the trasnfected cells. Results Puro screening lentivirus yielded the highest cell transfection efficiency,
which exceeded 90%, followed by Roche reagent and then by lipofectamine 2000. Flow cytometry and CCK-8 test showed that
the cytotoxicity was the mildest with Roche reagent, moderate with lentivirus, and severe with lipofectamine 2000. The cells
trasnfected with lentivirus showed a significantly lower RIP140 expression than cells trasnfected with lipofectamine 2000 and
Roche reagent (P<0.05). Conclusion In Kupffer cells, lentivirus-mediated transfection, as compared with the other two
trasnfection reagents, can achieve good transfection efficiency with a relativelty low cytotoxicity, and allows for better
controllability and stability of the trasnfectiion conditions.