Reporter and over-expression plasmids

A novel Isl1 cardiac enhancer was identified, using comparative genomic analysis (genomes of chick, rat, zebrafish and human were used). The enhancer was cloned into previously described pTK vectors that contain a minimal promoter from the HSV thymidine kinase gene upstream of GFP/RFP, and were shown to be permissive for enhancer activity in chick embryos (Uchikawa et al., 2004). Control vectors for broad expression within the embryo, the reporter plasmids pCAGG-GFP/RFP, which were previously described (Nathan et al., 2008), were injected. The mouse Nkx2-5 cardiac enhancer was obtained from Prof. Eric Olson (Lien et al., 1999) and subcloned into the pTK vector for chick expression. To study the relationship between the hemangioblast and the cardiac mesoderm, we obtained a hemangioblast enhancer based on the chick Cerberus gene, that drives expression of GFP (Teixeira et al., 2011). For ectopic expression of Nkx2.5 and Tal1, chick coding sequences were cloned into pCAGG-IRES-GFP, which drives ubiquitous expression in every avian cell (Megason and McMahon, 2002).