FIGURE 1

Transduction of hES cells by modified HIV-1-based SIN18 vectors. (A) Schematic representation of the HIV-1 SIN18 vectors. The vector pRLLSIN18.hPGK.EGFP, which harbors EGFP under the control of the hPGK promoter, was modified to make pSIN18.cPPT.hEF1.EGFP.WPRE by (1) insertion of WPRE downstream of the reporter gene, (2) replacement of the hPGK promoter by the hEF1 promoter, and (3) re-introduction of cPPT upstream of the promoter. (B) FACS analysis of hES cells 7 days after transduction with the nonmodified vector (middle panel) and the modified vector using an improved transduction protocol (right panel). The level of spontaneous differentiation was determined by analysis of the percentage of cells that were immunoreactive with GCTM2 antibody. The percentages of cells are indicated in each quadrant together with the mean fluorescence intensity (in parentheses) of EGFP expression. The upper and lower right quadrants represent the undifferentiated and differentiated transduced hES cells, respectively. The dot-plot analyses show the results of one representative experiment out of three experiments.