Fig. 5

Tg(–3.5ubi:cre^ERt2;cmlc2-EGFP) provides 4-OHT-inducible ubiquitous Cre activity. Asterisks indicate cmlc2-EGFP expression in the heart, which segregates with the ubi:cre^ERt2 transgene. (A) Schematic of the ubi:cre^ERt2 transgene and lmo2:lox-dsRed-lox-EGFP (lmo2:Switch) reporter cross shown in panels (B-I). Black arrows indicate transcription start positions, yellow boxes are non-coding 52 and 32 UTR/polyA sequences, blue arrow heads indicate loxP sites. (B-I) Images depict the posterior region of live ubi:cre^ERt2; lmo2:Switch double-positive zebrafish embryos at approximately 24 hpf, with insets showing cmlc2:EGFP expression to confirm presence of both transgenes. Embryos were induced at shield stage (6 hpf) with 5 μM 4-OHT (B-E) or ethanol (EtOH) carrier (F-I). mCherry expression marks lmo2:Switch-expressing cells in endothelial cell populations of the tail and head (C,G), whereas EGFP reveals specific 4-OHT dependent loxP cassette excision from lmo2:Switch by Cre (D,E), which did not occur in ethanol controls (H,I). (J) Schematic of the ubi:cre^ERt2 transgene and Tg(eab2:[EGFP-T-mCherry]) (FlEX) reporter cross shown in K and L. Note that FlEX is driven by a compound ef1α:β-actin2 promoter fragment which drives a bi-directional Terminator (T)-separated EGFP/mCherry cassette that inverts to enable mCherry expression after Cre-mediated recombination (Boniface et al., 2009). (K,L) Live ubi:cre^ERt2; FlEX double-positive zebrafish embryos at approximately 3 dpf. The embryo on top was treated with 5 μM 4-OHT at shield stage, whereas the embryo at the bottom was treated with ethanol carrier control. EGFP fluorescence indicates default FlEX reporter expression (K), whereas mCherry fluorescence (L) reveals successful Cre-mediated loxP recombination, which inverts the EGFP-T-mCherry cassette in FlEX and triggers mCherry expression.