To monitor therapeutic transgene expression, we developed fusion genes of
enhanced green fluorescent protein (EGFP) with two different
prodrug-activating
enzyme genes: herpes simplex virus type 1
thymidine kinase (HSV-tk) and rabbit
cytochrome P450 4B1 (
cyp4b1). Expression of the resulting fusion
proteins, TK-EGFP and 4B1-EGFP, rendered transduced human and rodent
glioma cells sensitive to cytotoxic treatment with the corresponding
prodrugs ganciclovir and
4-ipomeanol.
Ganciclovir and
4-ipomeanol sensitivity was comparable with that achieved with the native HSV-TK and
CYP4B1 proteins. As shown by fluorescence microscopy, TK-EGFP was expressed predominantly intranuclearly, whereas 4B1-EGFP was detectable in the cytoplasm, thereby displaying the orthotopic subcellular distribution of the corresponding native
enzymes. The fluorescence intensity correlated well with the corresponding
prodrug sensitivity, as shown by fluorescence-activated cell sorter analysis. EGFP expression was also used for the selection of stably HSV-tk-transduced cells by flow cytometric cell sorting. Resulting cell populations showed a homogeneity of fluorescence intensity similar to single-cell clones after
antibiotic selection. In conclusion, tk-egfp and 4b1-egfp fusion genes are valuable tools for monitoring
prodrug-activating gene therapy in living cells. EGFP fusion genes/
proteins provide a simple and reproducible means for the detection, selection, and characterization of cells expressing
enzyme genes for
prodrug activation.