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SL Semple-Rowland, GE Fuchs, JE Coleman; Design and Function of a Self-contained, Tetracycline-inducible Transgene for use in Lentiviral-mediated Gene Delivery Systems . Invest. Ophthalmol. Vis. Sci. 2002;43(13):2889.
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Purpose: To design a self-contained, tetracycline (Tet)-inducible system that utilizes a transrepressor protein to maximize repression of Tet-regulated gene transcription in the absence of antibiotic. Methods: A bicistronic transcriptional unit under the control of a chicken ß-actin (CBA) promoter was constructed containing the genes encoding the reversed TetR-VP16 transactivator (rtTA) and the modified transrepressor TetR(B/E)-KRAB (tTR; provided by W. Hillen) joined by the polio IRES. This transgene was placed upstream of a second, independent transcriptional unit that consisted of the gene encoding destabilized enhanced GFP (d2EGFP) under the control of a Tet responsive, minimal CMV promoter (TRE). The function of the resulting vector (CBA-rtTA-IRES-tTR-TRE-d2EGFP) and three control vectors (TRE-d2EGFP, CBA-tTR-TRE-d2EGFP, CMV-EGFP) was examined in DF1 cell cultures grown in DMEM containing Tet-free FBS. Cultures were transfected with 1.4 µg DNA and expression of GFP was examined in either the presence or absence of 10 µg/ml doxycycline (DOX). Levels of GFP expression were estimated by counting GFP-positive (GFP+) cells every 12 hours over the course of a 60-hour period. The number of GFP+ cells in cultures transfected with TRE-d2EGFP was the baseline to which the expression of all other vectors was compared. Results: In the absence of DOX, the number of GFP+ cells in cultures transfected with either CBA-rtTA-IRES-tTR-TRE-d2EGFP or CBA-tTR-TRE-d2EGFP was reduced by 84% compared to baseline. Addition of DOX to cultures transfected with CBA-rtTA-IRES-tTR-TRE-d2EGFP produced a rapid (within 12-24 hours), 24-fold increase in the number of GFP+ cells relative to cultures without DOX, a value that was comparable to that observed in cultures transfected with CMV-EGFP. Addition of DOX to cultures transfected with CBA-tTR-TRE-d2EGFP significantly increased the number of GFP+ cells, but the increase was significantly below baseline. Addition of DOX to cultures transfected with TRE-d2EGFP or CMV-EGFP did not significantly alter the number of GFP+ cells in these cultures. Conclusion: Inclusion of the tTR transrepressor in our Tet-inducible system significantly reduced basal levels of expression of the TRE-d2EGFP transcriptional unit in the absence of DOX. Current studies are aimed at packaging the CBA-rtTA-IRES-tTR-TRE-d2EGFP transgene into lentivirus and examining its expression in vivo. Successful regulation of this transgene in vivo will permit studies of the relationship between disease progression and therapy efficacy.
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