Enhancer detection in the zebrafish using pseudotyped murine retroviruses.
Vectors based on murine retroviruses are among the most efficient means to insert reporter constructs into the context of a vertebrate chromosome with the aim to visualize cis-regulatory information available to a basal promoter at the site of insertion. In combination with using the zebrafish embryo as a readout for the activity of regulatory elements, enhancer detection becomes a powerful technique for gene discovery and for the mapping of the extent of regulatory domains in a vertebrate genome. Our laboratory has performed the only large-scale enhancer detection screen to date in any vertebrate and we describe in this paper the methods we developed to generate viral particles, to insert reporter constructs into the zebrafish germ line, the screening of detection events in heterozygous F1 embryos, and the isolation of genomic sequence flanking the inserted vector for the purpose of genomic mapping. Given sufficient scale, the technology described here can be used to obtain cis-regulatory information across the entire zebrafish genome for any given basal promoter.
Laplante
Mary
M
Sars Centre for Marine Molecular Biology, University of Bergen, High Technology Centre, Thormoehlensgate 55, 5008 Bergen, Norway.
Kikuta
Hiroshi
H
König
Melanie
M
Becker
Thomas S
TS
D000818:Animals; D030801:Animals, Genetically Modified / Q000235:genetics*; D003001:Cloning, Molecular; D019295:Computational Biology; D004742:Enhancer Elements, Genetic; D017930:Genes, Reporter; D005818:Genetic Engineering / Q000295:instrumentation / Q000379:methods*; D005822:Genetic Vectors; D023281:Genomics / Q000379:methods; D051379:Mice; D012190:Retroviridae / Q000235:genetics*; D017422:Sequence Analysis, DNA; D014162:Transfection / Q000295:instrumentation / Q000379:methods; D015027:Zebrafish / Q000196:embryology / Q000235:genetics*