title: 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.
authors.affiliation: Brain and Mind Research Institute, Sydney Medical School, University of Sydney, 100 Mallet Street, Camperdown 2050, Australia.
authors.forename: Minaka
authors.initials: M
authors.lastname: Ishibashi
authors.forename: Alejandro S
authors.initials: AS
authors.lastname: Mechaly
authors.forename: Thomas S
authors.initials: TS
authors.lastname: Becker
authors.forename: Silke
authors.initials: S
authors.lastname: Rinkwitz
mesh_terms: D000818:Animals; D030801:Animals, Genetically Modified; D004251:DNA Transposable Elements; D004742:Enhancer Elements, Genetic; D005260:Female; D018703:Founder Effect; D018507:Gene Expression Regulation, Developmental; D017930:Genes, Reporter; D056426:Genetic Loci; D005822:Genetic Vectors; D016678:Genome; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D008297:Male; D008845:Microinjections; D019076:Transgenes; D015027:Zebrafish / Q000235:genetics* / Q000254:growth & development; D015053:Zygote / Q000254:growth & development / Q000378:metabolism
chemical_list: D004251:DNA Transposable Elements; D049452:Green Fluorescent Proteins
keywords: Enhancer; Gene regulation; Human genome; Mammalian genome; Reporter gene expression; Vertebrate genome; Zebrafish transgenesis