In vivo characterisation of disease-associated cis-regulatory variants by dual fluorescence reporter transgenic analysis in zebrafish. Schematic representation of the assay pipeline (modified from [20]). Both alleles (wild type and mutant) of potential disease-associated CREs are cloned in a construct with a reporter cassette of choice, to create the cis-element-reporter cassette flanked by Tol2 sites. Gata2-promoter (g2 prom) derived from mouse genome is included in the CRE-reporter constructs to serve as a minimal promoter in the assay. Co-injection of reporter constructs containing wild type (Wt) and mutant (Mut) versions of the CRE with Tol2 transposase-encoding RNA into early zebrafish embryos results in independent integration of the reporter cassettes. Transgenic founders (F0) are bred to establish transgenic lines. Expression patterns are examined in fish of F1 or later generations. Differences between Wt and Mut elements can be compared directly in the same fish using the GFP and mCherry fluorescent reporters.