Functional coexpression of HSV-1 thymidine kinase and green fluorescent protein: Implications for noninvasive imaging of transgene expression Journal Article
| Authors: | Jacobs, A.; Dubrovin, M.; Hewett, J.; Sena-Esteves, M.; Tan, C. W.; Slack, M.; Sadelain, M.; Breakefield, X. O.; Tjuvajev, J. G. |
| Article Title: | Functional coexpression of HSV-1 thymidine kinase and green fluorescent protein: Implications for noninvasive imaging of transgene expression |
| Abstract: | Current gene therapy technology is limited by the paucity of methodology for determining the location and magnitude of therapeutic transgene expression in vivo. We describe and validate a paradigm for monitoring therapeutic transgene expression by noninvasive imaging of the herpes simplex virus type 1 thymidine kinase (HSV-1-tk) marker gene expression. To test proportional coexpression of therapeutic and marker genes, a model fusion gene comprising green fluorescent protein (gfp) and HSV-1-tk genes was generated (tkgfp gene) and assessed for the functional coexpression of the gene product, TKGFP fusion protein, in rat 9L gliosarcoma, RG2 glioma, and W256 carcinoma cells. Analysis of the TKGFP protein demonstrated that it can serve as a therapeutic gene by rendering tkgfp transduced cells sensitive to ganciclovir or as a screening marker useful for identifying transduced cells by fluorescence microscopy or fluorescence-activated cell sorting (FACS). TK and GFP activities in the TKGFP fusion protein were similar to corresponding wild-type proteins and accumulation of the HSV-1-tk-specific radiolabeled substrate, 2′-fluoro-2′-deoxy-1β-D-arabinofuranosyl-5-iodo-uracil (FIAU), in stability transduced clones correlated with gfp-fluorescence intensity over a wide range of expression levels. The tkgfp fusion gene itself may be useful in developing novel cancer gene therapy approaches. Valuable information about the efficiency of gene transfer and expression could be obtained by non-invasive imaging of tkgfp expression with FIAU and clinical imaging devices (gamma camera, positron-emission tomography [PET], single photon emission computed tomography [SPECT]), and/or direct visualization of gfp expression in situ by fluorescence microscopy or endoscopy. |
| Keywords: | promoter region; genetics; dose response; flow cytometry; methodology; animal; metabolism; animals; green fluorescent protein; enzymology; dose-response relationship, drug; tumor cells, cultured; photoprotein; genetic transduction; luminescent proteins; transduction, genetic; molecular cloning; cloning, molecular; blotting, western; hybrid protein; recombinant fusion proteins; cell culture; rat; western blotting; gene therapy; transgene; drug derivative; uracil arabinoside; arabinofuranosyluracil; imaging; green fluorescent proteins; fluorescence microscopy; microscopy, fluorescence; fialuridine; thymidine kinase; herpesvirus 1, human; rats; antivirus agent; herpes simplex virus 1; cell separation; ganciclovir; transgenes; complementary dna; retrovirus; retroviridae; dna, complementary; antiviral agents; cancer gene therapy; fiau; promoter regions (genetics); article; fusion genes |
| Journal Title: | NeoPlasia |
| Volume: | 1 |
| Issue: | 2 |
| ISSN: | 1522-8002 |
| Publisher: | Elsevier Science Inc. |
| Date Published: | 1999-06-01 |
| Start Page: | 154 |
| End Page: | 161 |
| Language: | English |
| PUBMED: | 10933050 |
| PROVIDER: | scopus |
| PMCID: | PMC1508134 |
| DOI: | 10.1038/sj.neo.7900007 |
| DOI/URL: | |
| Notes: | Article -- Export Date: 16 August 2016 -- Source: Scopus |
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