Figure 5.

The March-I v2 promoter is inactive in non-APCs. Epithelial (HeLa) cells, kidney (HEK-293) cells, mouse embryonic fibroblasts, and BM DCs were transduced with lentivirus encoding GFP using the CMV promoter, the March-I v2 (−663 to +125) “forward” promoter, or a negative “control promoter.” Expression of GFP, puromycin, and GAPDH mRNA was determined by RT-PCR. Data using HEK-293 cells, HeLa cells, BM DCs, and spleen B cells were normalized to the amount of puromycin mRNA present in the sample, and the control was the promoterless lentivirus. Data using MEFs were normalized to the amount of GAPDH mRNA present in the sample, and the control was lentivirus with the March-I v2 (−663 to +125) promoter inserted into the expression vector in the backward orientation. In each sample, the amount of GPF mRNA was expressed relative to the amount of mRNA detected using the CMV promoter construct (arbitrarily designated a value of 1). Control experiments using RNA from MEFs, HEK-293 cells, HeLa cells, or BM DCs that were not reverse transcribed revealed negligible contamination of cDNA with host cell genomic DNA or lentivirus DNA. Spleen B-cell cDNA did contain contaminating lentivirus DNA, and therefore RT-PCR data were calculated as 2^ΔCt (ΔCt = Ct_{plus RT} − Ct_{minus RT}). The results shown are the average (error bars represent S.D.) of three independent experiments. The expression of GFP driven by the March-I v2 promoter in each cell type was compared with that using the control promoter. *, p < 0.05; ns, not significant.