Tol2 transposon‐mediated enhancer trap to identify developmentally regulated zebrafish genes in vivo

Abstract We have used the Tol2 transposable element to design and perform effective enhancer trapping in zebrafish. Modified transposon DNA and transposase RNA were delivered into zebrafish embryos by microinjection to produce heritable insertions in the zebrafish genome. The enhancer trap construct carries the EGFP gene controlled by a partial epithelial promoter from the keratin8 gene. Enhanced green fluorescent protein (EGFP) is used as a marker to select F 1 transgenic fish and as a reporter to trap enhancers. We have isolated 28 transgenic lines that were derived from the 37 GFP‐positive F 0 founders and displayed various specific EGFP expression patterns in addition to basal expression from the modified keratin 8 promoter. Analyses of expression by whole‐mount RNA in situ hybridization demonstrated that these patterns could recapitulate the expression of the tagged genes to a variable extent and, therefore, confirmed that our construct worked effectively as an enhancer trap. Transgenic offspring from the 37 F 0 EGFP‐positive founders have been genetically analyzed up to the F 2 generation. Flanking sequences from 65 separate transposon insertion sites were identified by thermal asymmetric interlaced polymerase chain reaction. Injection of the transposase RNA into transgenic embryos induced remobilization of genomic Tol2 copies producing novel insertions including some in the germ line. The approach has great potential for developmental and anatomical studies of teleosts. Developmental Dynamics 231:449–459, 2004. © 2004 Wiley‐Liss, Inc.