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. 2001 Aug 21;98(19):10775–10780. doi: 10.1073/pnas.121186498

Figure 2.

Figure 2

Analysis of the Tet-On TA (rtTA-M2-alt) in adult flies. (A) Dose curve for target gene induction. Double transgenic flies were maintained on dox-free food (0) or food supplemented with 1, 10, 100, or 1,000 μg/ml of dox for 24 h. Whole fly extracts were analyzed on Western blots using a luciferase mAb. Extracts also were made from control flies carrying two copies of the luciferase target transgene (−), without a TA transgene, and not fed antibiotic. (B) Induction kinetics of flies fed dox at 10 μg/ml. Double transgenic flies were frozen immediately (0), 6, 12, 24, 36, or 48 h after being placed on dox-containing food (10 μg/ml), extracts were prepared, and Western blot analysis was done with the luciferase antibody. Extracts also were made from control flies carrying two copies of the target transgene without a TA (−), and not fed antibiotic. (C) Induction kinetics of flies fed dox at 1 mg/ml. Double transgenic flies were frozen immediately (0), 1, 3, 6, 12, 24, or 48 h after being placed on dox-containing food, extracts were prepared, and Western blot analysis was done with the luciferase antibody. Extracts also were made from control flies carrying two copies of the target transgene without a TA (−), and not fed antibiotic. (D) Induction kinetics of flies injected with dox. Double transgenic flies were injected with ≈1 nl of a 10 mg/ml dox solution, and extracts were prepared 30 min, 1, 3, 6, or 12 h after injection. Extracts also were made from flies injected with PBS (Mock), or single transgenic flies missing the TA (−) and uninjected. Levels of induction: (E) Effect of TA gene dosage on target gene expression levels. Flies carrying one or two copies of the rtTA-M2-alt and two copies of a luciferase target transgene were frozen at 0, 12, or 24 h after being placed on dox-containing food (1 mg/ml). Extracts also were made from control flies containing two copies of the luciferase target transgene without a TA source (−) and not fed antibiotic. Western blot analysis was done with the luciferase antibody. (G) Quantitative analysis using in vitro luciferase assays. Twelve hours after the start of drug feeding, extracts from flies containing one (rtTA-M2-alt/CyO) or two (rtTA-M-alt/rtTA-M2-alt) copies of the TA were assayed for luciferase activity. (F) Estimate of the absolute amount of luciferase protein that is induced. Western blot analysis of extracts using a luciferase-specific mAb. Twenty micrograms of total protein from double transgenic flies fed antibiotic (+Dox), or not (−Dox), was compared with extracts from wild-type flies supplemented with known quantities (1, 2.5, or 5 ng) of purified luciferase protein.