Fig. 4

The inducible dominant mutagenesis is validated in a more focused genetic screen for cardiovascular mutants. (A) A modified pIDM vector (ubi-pIDM) for testing Dox-induced GFP expression. GFP is located downstream to TRE3G promoter, rtTA and transgenic reporter RFP are driven by the ubi promoter, of which rtTA and RFP are linked by T2A, and the ubi-rtTA-T2A-RFP cassette is flanked by a pair of insulators. (B–E) Transgenic reporter RFP of F₁ embryos were expressed in both control (B) and Dox-induced (C) embryos. GFP was hardly expressed in control group (D) but highly induced by Dox (E). (F) Schematic of the screen strategy for cardiovascular mutants. F₀ founders were crossed with WT to establish F₁ transgenic zebrafish. The F₁ transgenic fish with RFP was crossed with WT to get F₂ embryos. The F₂ embryos were divided into two groups, one treated with Dox at 6–12 hpf for screening cardiovascular defects, and another one for raising F₂ embryos into adults. (G) The ubi-pIDM vector insertion is located at 625 bp upstream to the 5 ‘UTR of dnajb5 in #84 line. (H) RT-PCR showed that dnajb5 mRNA was 3 times more after Dox induction. *p < 0.05. (I–L) #84Tg had cardiovascular defects (J) compared with WT siblings (I) at 48 hpf after Dox induction. Embryos injected with dnajb5 mRNA (0.1ng) (L) mimicked the phenotypes of Dox treated #84Tg embryos. Lower right numbers show phenotypical embryos out of total embryos analyzed. (M–R) Cartoon shows the ubi-pIDM vector position in the 3 ‘UTR of proza in Chr. 3 in #13 line. The proza 5′UTR sequence was used to drive EGFP expression, the start code ATG of proza is in red and underlined, and the prozaMO sequence is underlined (M). The proza:EGFP reporter was expressed in control embryos (upper panel) that was inhibited by prozaMO (4 ng) (lower panel) at 24 hpf. (O–R) #13 transgenic embryos (#13Tg) showed no blood circulation and abnormal tail development (P) compared with #13 control siblings (O) after Dox induction. Morphant embryos with 4 ng prozaMO showed developmental defects (R) similar to that in #13Tg embryos (P) at 48 hpf.