Figure 4

Characterizing rtTA-M2 transgene expression from homozygous ROSA26- rtTA-M2-1089/1088 transgenic rats (single integration). A homozygous rtTA-M2 transgenic rat line (ROSA26-rtTA-M2-1089/1099; renamed ROSA-rtTA-M2) was established by breeding F1 Tg1088 (male) and F1 Tg1089 (female), which each exhibited a single common integration of the ROSA26-rtTA-M2 transgene. A. Doxycycline dose response analysis. Fibroblasts from ROSA-rtTA-M2 F2 progeny were transduced with Lenti-TetO₇/CMV-EGFP and cultured in medium containing different concentrations of Dox (0 to 1000 ng/ml). Western blot analyses were used to evaluate EGFP expression. The blot was stripped and reprobed with β-actin antibody as a loading control. B-E. rtTA-M2 gene was stably transmitted. Fibroblasts from the F5 generation of homozygous ROSA-rtTA-M2 rats were transduced with Lenti-TetO₇/CMV-EGFP and cultured in medium without (B and C) or with (D and E) Dox (500 ng/ml). EGFP expression was observed by immunocytochemistry 48 hours post-transduction (Bar = 20 μm). F. Real time-PCR analysis to determine tissue distribution of rtTA-M2 expression from ROSA-rtTA-M2 rats. rtTA-M2 expression in each tissue is presented relative to the rtTA-M2 expression in the testis. The whiskers represent the standard errors. β-actin was used as an internal control for each tissue.