title: Harnessing mobile genetic elements to explore gene regulation.
abstract: Sequences that regulate expression of a gene incisbut are located at large distances along the DNA from the gene, as found with most developmentally regulated genes in higher vertebrates, are difficult to identify if those sequences are not conserved across species. Mutating suspected gene-regulatory sequences to alter expression then becomes a hit-or-miss affair. The relaxed specificity of transposon insertions offers an opportunity to develop alternate strategies, to scan in an unbiased manner, pieces of chromosomal DNA cloned in BACs for transcription enhancing elements. This article illustrates how insertions of Tn10 with enhancer-traps into BAC DNA containing the gene, and its germ-line expression in zebrafish, have identified distal regulatory elements functionally. Transposition of Tn10 first introduces the enhancer-trap with a loxP site randomly into BAC DNA. Cre-recombination between the inserted loxP and the loxP endogenous to a BAC-end positions the enhancer-trap to the newly created truncated end of BAC DNA. The procedure generates a library of integration-ready enhancer-trap BACs with progressive truncations from an end in a single experiment. Individual enhancer-trap BACs from the library can be evaluated functionally in zebrafish or mice. Furthermore, the ability to readily alter sequences in a small transposon plasmid containing a regulatory domain of the gene allows re-introduction of altered parts of a BAC back into itself. It serves as a useful strategy to functionally dissect multiple discontinuous regulatory domains of a gene quickly. These methodologies have been successfully used in identifying novel regulatory domains of the Amyloid Precursor Protein (appb) gene in zebrafish, and provided important clues for regulation of the gene in humans.
authors: Julius L. Chambers Biomedical/ Biotechnology Research Institute and Department of Chemistry; North Carolina Central University; Durham, NC USA.
authors: Leighcraft A
authors: LA
authors: Shakes
authors: Julius L. Chambers Biomedical/ Biotechnology Research Institute and Department of Chemistry; North Carolina Central University; Durham, NC USA.
authors: Hope M
authors: HM
authors: Wolf
authors: Julius L. Chambers Biomedical/ Biotechnology Research Institute and Department of Chemistry; North Carolina Central University; Durham, NC USA.
authors: Derek C
authors: DC
authors: Norford
authors: Julius L. Chambers Biomedical/ Biotechnology Research Institute and Department of Chemistry; North Carolina Central University; Durham, NC USA.
authors: Delores J
authors: DJ
authors: Grant
authors: Julius L. Chambers Biomedical/ Biotechnology Research Institute and Department of Chemistry; North Carolina Central University; Durham, NC USA.
authors: Pradeep K
authors: PK
authors: Chatterjee
keywords: amyloid precursor protein gene regulation; enhancer-trap BACs; gene regulation by discontinuous DNA domains; long-range gene regulation by enhancer-trap BACs; new technology for BAC modification; regulation of APP gene in humans; regulation of appb gene in zebrafish; zebrafish model for gene regulation in humans; zebrafish transgenesis