Figure 3

A) Packaging cell expression. Packaging cells were transfected with equal amounts of pC-GFP, pC-cGreen and pC-neerGc retroviral vector plasmids. GFP expression was quantified in single cells at 24 hours by flow cytometry. B) Expression levels in infected cells. At 18 hours post-transfection, conditioned medium was harvested from the packaging cells and used to infect human umbilical vein cells (HUVEC). GFP expression was quantified in single cells at 48 hours post-infection by flow cytometry. pC-GFP infected cells expresses strong GFP levels, while neither the pC-cGreen nor the pC-neerGc retroviral vector produced detectable viral infection. ND: none detected. C) A high expression retroviral vector using in vitro transcribed genomes. Early passage primary endothelial cells (HUVEC) were infected for 24 hours using serial dilutions of virus generated by the pC-GFP, pT-GFP, pT-cGreen and pT-neerGc vectors. The GFP expression level of individual cells harboring single viral integrations was measured by flow cytometry at 48 hours post-infection. GFP expression in the proviruses driven by the optimized CMV promoter/intron/pA cassette (pT-cGreen, pT-neerGc) is substantially higher than that obtained by LTR-promoter dependent expression (pC-GFP, pT-GFP).