Generation of miR30-A1 mice. (A) The 293T cells expressing FLAG-tagged mouse A1-a were transiently transfected with graded doses of the pEGFP-miR30-A1 vector that targets all A1 isoforms. The GFP⁺ cells were FACS-sorted, lysed, and extracts subjected to Western blot analysis using a FLAG-specific antibody and anti-GAPDH, to compare loading. (B) The 293T cells were transiently transfected with 2 μg of pGL2-Bim 0.8 kbp reporter plasmid, driving expression of the luciferase gene under the control of the first 800 bp of the Bim gene promoter plus graded doses of pEGFP-miR30-FF (0.5, 1.0, 2.0, or 4.0 μg), targeting firefly luciferase. At 24 hours after transfection, cells were lysed and a luciferase reporter assay performed. Bars represent mean of triplicates ± SD (n = 2). (C) Schematic representation of plasmids used for mouse transgenesis. (D) Transgenic expression of Venus in peripheral blood leukocytes of F1 offspring of PCR⁺ founders was monitored by flow cytometric analysis. Percentages given refer to Venus⁺ cells. (E) Transgenic eGFP expression was quantified in peripheral blood leukocytes of single-transgenic tTA, TREA1.1, TREA1.2, and DT mice. Percentages given refer to eGFP⁺ cells. (F) Transgene expression in peripheral blood of double-transgenic mice was monitored before and during doxycycline application in the drinking water by flow cytometric analysis. Percentages given refer to eGFP^+(low) and eGFP^++(high) cells at the beginning of doxycycline treatment, day 0, or after 19 days. (G) eGFP expression in DT mice was followed in the peripheral blood over an observation period of 200 days and analyzed by flow cytometry. Data are mean of n = 4 animals ± SEM.