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Context dependent function of APPb enhancer identified using enhancer trap-containing BACs as transgenes in zebrafish.

Nucleic Acids Res.2008 Nov;36(19):6237-48. Epub 2008 Oct 01
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摘要


An enhancer within intron 1 of the amyloid precursor protein gene (APPb) of zebrafish is identified functionally using a novel approach. Bacterial artificial chromosomes (BACs) were retrofitted with enhancer traps, and expressed as transgenes in zebrafish. Expression from both transient assays and stable lines were used for analysis. Although the enhancer was active in specific nonneural cells of the notochord when placed with APPb gene promoter proximal elements its function was restricted to, and absolutely required for, specific expression in neurons when juxtaposed with additional far-upstream promoter elements of the gene. We demonstrate that expression of green fluorescent protein fluorescence resembling the tissue distribution of APPb mRNA requires both the intron 1 enhancer and approximately 28 kb of DNA upstream of the gene. The results indicate that tissue-specificity of an isolated enhancer may be quite different from that in the context of its own gene. Using this enhancer and upstream sequence, polymorphic variants of APPb can now more closely recapitulate the endogenous pattern and regulation of APPb expression in animal models for Alzheimer's disease. The methodology should help functionally map multiple noncontiguous regulatory elements in BACs with or without gene-coding sequences.

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