Lentiviral Engineered 3T3 Cells Expressing p65-DsRed

We previously used a lentiviral system to create p65^−/− mouse fibroblast (3T3) cells expressing the fluorescent fusion protein p65-DsRed under control of the endogenous mouse p65 promoter. ⁶ Overexpression of p65 can cause unusual NF-KB activation, we therefore cloned the 1.5 kb upstream of the relA gene into the lentiviral construct to control p65-FP expression, and used the resulting construct to infect relA knockout 3T3 cells. These cells exhibited a normal response to TNF at the population level. To relieve a bottleneck in image processing in this study, we also infected the cells with a lentivirus containing the nuclear marker H2B-GFP driven by the human Ubiquitin C promoter. After cloning, the cells were frozen and newly thawed cells were used for each experiment to prevent 3T3 cells transforming and to minimize heterogeneity. A correlation between p65-DsRed levels and cell activation was not observed.