Using zebrafish transgenesis to test human genomic sequences for specific enhancer activity

Abstract

We detail an approach for the identification of human tissue-specific transcriptional enhancers involving three steps: delineation of search space around a locus or target gene, in silico identification and size definition of putative candidate sequences, and testing through several independent genomic insertions in a transgenic zebrafish reporter assay. Candidate sequences are defined through evolutionary conservation, transcription factor binding and chromatin marks (e.g. ENCODE data) and are amplified from genomic DNA, cloned into basal promoter:fluorescent protein reporter vectors based on the Tol2 transposon system and are microinjected into fertilized zebrafish eggs. After raising injected founders to sexual maturity, fluorescent screening identifies positive founder fish whose offspring undergo a detailed expression analysis to determine tissue specificity and reproducibility of specific enhancers.