Figure 4. Sequential transgene activation and inactivation using lox and rox (TAILOR).

(A) ubb:lox-stop-lox-rox-Nuc-mCherry-stop-rox-eGFP; ubb-CreER^T2; ubb-Dre triple transgenic fish were subjected to treatment with 4-OHT beginning at 24 hpf, followed by removal and replacement with RU486 at 48 hpf. The untreated triple transgenic embryos (24 hpf) showed no transgene-specific fluorescence besides that provided by the ocular mCherry, ocular eCFP and cardiac eGFP markers (Fig. 4A a1, a2, a3, and a4). The effective induction of nuclear mCherry expression was observed following 24 hrs of 4-OHT treatment (Fig. 4A b1 and b2). Following staged treatment with RU486 initiated at 48 hpf and left in place for 24 hrs, effective activation of eGFP expression was observed (Fig. 4A c1 and c4). Scale bar: 200 µm. (B) Quantification of relative numbers of intestinal, liver and pancreatic cells expressing mCherry (red), eGFP (green) or neither (blue) at 7 days following sequential 4OHT and RU486 exposure as above. Note high fraction of cells undergoing sequential CreER^T2-mediated activation and DrePR-mediated inactivation of mCherry expression, as indicated by eGFP expression. Scale bar: 25 µm.