Colocalization studies using recombineered BAC vectors. (A) Examples of stable, single-copy transgenic lines obtained using the described protocol. Lateral views of whole zebrafish embryos at 4-7 dpf. BAC clone IDs and predicted zebrafish genomic DNA insert sizes are indicated in the top right corner. (B) Dorsal view of the head of an sp7:gfp (Spoorendonk et al., 2008) transgenic zebrafish embryo at 5 dpf injected with col2a1a:mCherry BAC DNA and tol2 transposase mRNA. Note the colocalization of GFP and mCherry in at least one cell (arrow). (C). Lateral view of the trunk of a kdrl:mCherry-CAAX (Hogan et al., 2009) transgenic embryo at 48 hpf injected with a flt4:yfp (citrine) BAC and tol2 transposase mRNA. Individual YFP⁺ endothelial cells (yellow) are detectable within the posterior caudal vein and intersegmental vessels.

Comparison of transgenic reporter mRNA expression with endogenous mRNA expression. Whole-mount in situ hybridization with antisense RNA probes to (A) cdh5 or (B) gfp/yfp in Tg(cdh5:gal4)^mu101; Tg(uas:gfp)^nkuasgfp1a double-transgenic embryos (Bussmann et al., 2011) and to (C) cxcl12b or (D) gfp/yfp in Tg(cxcl12b:yfp)^mu102 transgenic embryos. The expression patterns of the transgenic reporter construct and the endogenous gene were found to be identical. We note that the expression levels of the cxcl12b:yfp reporter were similar to those of the endogenous cxcl12b gene, whereas the expression levels of the cdh5:gal4, uas:gfp reporter were significantly higher than those of the endogenous cdh5 gene. No expression differences of the endogenous genes were detected in transgenic versus non-transgenic embryos.