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. Author manuscript; available in PMC: 2010 Aug 1.
Published in final edited form as: Gene Ther. 2009 Oct 15;17(2):238–249. doi: 10.1038/gt.2009.131

Figure 1. GFP expression in DHFR-GFP transduced hESCs.

Figure 1

(a) DHFR-GFP lentivirus vector schematics. The human EF1-α promoter regulates transcription of Tyr22-DHFR and/or GFP, along with an internal ribosomal entry site (ires) between the two coding sequences (EFDIG) or as a genetic fusion (DL2G). In a two-promoter configuration (REDPeG), EF1-α regulates Tyr22-DHFR and the human PGK promoter regulates GFP expression. Other features (described in Materials and Methods) include the HIV-1 R, U5, self-inactivating U3, and rev response element (rre), and the post-transcriptional regulatory element from the woodchuck hepatitis virus ([w2]wpre). (b) GFP expression was evaluated by fluorescence microscopy of the lentivirus transduced hES cell line H9 4 days after transduction. All images (10–20× objective) were captured at the same exposure time and modified identically in Photoshop CS2 version 9 (Brightness altered to −60, contrast +35 then applied smart sharpen). (c) Flow cytometric evaluation of GFP expression at 4 weeks. Transduced cells were stained with PE-conjugated anti-SSEA-4 to assess co-expression of GFP and surface expression of SSEA-4 as a marker for undifferentiated hESCs.