FIG. 5.
Efficiency of EGFP marker loss in pICUT vectors. The two vectors schematically represented in panel C were constructed and used to make retroviral stocks by transient transfection. This virus was then used to transduce HT1080 cells. (A) Photographic record of EGFP expression in both transfected and transduced cells. (B) Results of EGFP analysis by FACS. For each cell type (transfected or transduced with either pGiresZN or pICUT-GZN vectors) the mean count for cell fluorescence was established and used to assess EGFP expression levels. For both vectors analyzed, the EGFP genes are expressed from a cytomegalovirus-based LTR promoter upon transfection and from a U3-based LTR upon transduction. To ensure normalization of transfection and transduction efficiencies between vectors, FACS analysis was performed on only the “gated” fluorescent-cell population. Titers of pGiresZN and pICUT-GZN for this experiment were 9 × 105 and 1 × 105 LFU/ml, respectively. This figure illustrates that upon transduction and relative to control, the EGFP expression from the pICUT vector is decreased.