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

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