Figure 1.
Tight control of reporter gene expression using the TRE-PminiCMV system. A) pTL is the backbone plasmid that only contains rtTA and tTS cDNAs. TRE/loxP-inducible GFP (pTLiG) and TRE/loxP-inducible luciferase (pTLiL) are plasmids used to test the Dox-inducibility of reporter gene expression. In pTLcG, the GFP reporter gene is constitutively driven by the PSV40 promoter. B) Examination of Dox-inducible GFP expression of pTLiG in HEK293 and CHO cells. HEK293 or CHO cells in 6-well plates were transfected with 1.0 μg of pTLiG or pTLcG plasmid. At 24 h after transfection, the cells were cultured further in a medium with or without Dox (1.0 μg/ml) for 24 h. GFP expression from transfected HEK293 or CHO cells was observed by fluorescence microscopy. C) Examination of Dox-inducible luciferase expression of pTLiL in HEK293 and CHO cells. HEK293 or CHO cells on 24-well plates were transfected with 0.5 μg of pTLiL or pTL plasmid. At 24 h after transfection, the cells were cultured further with or without Dox (1.0 μg/ml) for 24 h. Expression levels of luciferase were measured from HEK293 or CHO cells. Data show tight regulation of gene expression (<12% of leaky expression) in the absence of Dox, as compared with modified U6 promoters containing TRE sequence (see Supplemental Fig. S1) and robust induction in the presence of Dox (8- to 17-fold induction of luciferase reporter genes, depending on cell type).