Generation of Dusp6 Reporter Transgenic Zebrafish

A Dusp6 BAC clone was identified by PCR from BAC DNA pools as directed by manufacturers protocols (Genome Systems). A 10 Kb Kpn1 fragment was identified that contain parts of Exon 1 (441 bp) and approximately 9.5 Kb of upstream promoter sequence (see Figure 1A). This Kpn1 fragment was subcloned into the Kpn1 site of pSce1d2EGFP vector (pSce1 vector with d2EGFP cDNA cloned into the multiple cloning site). 20 pg of pDusp6:d2EGFP plasmid DNA was injected into the 1-cell embryo with I-Sce 1 (New England Biolabs, Ipswich, MA) restriction enzyme as described in [37 (link)]. These Founder F0 injected embryos were raised to adulthood and incrossed to identify transgenic founders. We identified 10 founder lines that expressed d2EGFP within regions of known FGF activity in the developing embryo. Four lines exhibited strong expression throughout development and were maintained. Tg(Dusp6:d2EGFP)^pt6 was used predominantly in this study.