In vitro and in vivo characterization of a dual-function green fluorescent protein--HSV1-thymidine kinase reporter gene driven by the human elongation factor 1 alpha promoter.
Toward the goal of monitoring activity of native mammalian promoters with molecular imaging techniques, we stably transfected DU145 prostate carcinoma cells with a fusion construct of enhanced green fluorescent protein (EGFP) and wild-type herpes simplex virus-1 thymidine kinase (HSV1-TK) as a reporter gene driven by the promoter for human elongation factor 1 alpha (EF-1 alpha-EGFP-TK). Using this model system, expression of EGFP was quantified by flow cytometry and fluorescence microscopy, while the HSV1-TK component of the reporter was quantified with 8-[3H]ganciclovir (8-[3H]GCV). As analyzed by flow cytometry, passage of EGFP-TK-DU145 transfected cells (ETK) in vitro resulted in populations of cells with high and low expression of EGFP over time. High and low ETK cells retained 23-fold and 5-fold more GCV, respectively, than control. While differences in uptake and retention of GCV corresponded to relative expression of the reporter gene in each subpopulation of cells as determined by both flow cytometry (EGFP) and quantitative RT-PCR, the correlation was not linear. Furthermore, in high ETK cells, net retention of various radiolabeled nucleoside analogues varied; the rank order was 8-[3H]GCV < 9-(4-fluoro-3-hydroxymethylbutyl)guanine ([18F]FHBG) approximately 8-[3H]penciclovir (8-[3H]PCV) < 2'-fluoro-2'-deoxy-5-iodouracil-beta-D-arabinofuranoside (2-[14C]FIAU). Xenograft tumors of ETK cells in vivo accumulated 2.5-fold more 8-[3H]GCV per gram of tissue and showed greater fluorescence from EGFP than control DU145 cells, demonstrating that the reporter gene functioned in vivo. These data extend previous reports by showing that a human promoter can be detected in vitro and in vivo with a dual-function reporter exploiting optical and radiotracer techniques.
Luker
Gary D
GD
Washington University School of Medicine, 510 S. Kingshighway Blvd, St. Louis, MO 63110, USA.
Luker
Kathryn E
KE
Sharma
Vijay
V
Pica
Christina M
CM
Dahlheimer
Julie L
JL
Ocheskey
Joe A
JA
Fahrner
Timothy J
TJ
Milbrandt
Jeffrey
J
Piwnica-Worms
David
D
D001483:Base Sequence; D017931:DNA Primers; D005434:Flow Cytometry / Q000379:methods; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins; D018259:Herpesvirus 1, Human / Q000201:enzymology / Q000235:genetics; D006801:Humans; D007700:Kinetics; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism*; D008297:Male; D008856:Microscopy, Fluorescence / Q000379:methods; D020648:Peptide Elongation Factor 1 / Q000235:genetics*; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic; D011471:Prostatic Neoplasms; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013937:Thymidine Kinase / Q000235:genetics / Q000378:metabolism*; D014162:Transfection; D014407:Tumor Cells, Cultured
D017931:DNA Primers; D008164:Luminescent Proteins; D020648:Peptide Elongation Factor 1; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D013937:Thymidine Kinase