Nucleic Acids Research Advance Access originally published online on October 1, 2008
Nucleic Acids Research 2008 36(19):6237-6248; doi:10.1093/nar/gkn628
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Nucleic Acids Research, 2008, Vol. 36, No. 19 6237-6248
© 2008 The Author(s)
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Gene Regulation, Chromatin and Epigenetics |
Context dependent function of APPb enhancer identified using enhancer trap-containing BACs as transgenes in zebrafish
1Julius L. Chambers Biomedical/Biotechnology Research Institute, 2Department of Chemistry, 3Department of Biology, North Carolina Central University, Durham, NC 27707 and 4Gene Expression and Genomics Unit, National Institute on Aging, NIH, Triad Technology Center, Baltimore, MD 21224, USA
*To whom correspondence should be addressed. Tel: +1 919 530 7017; Fax: +1 919 530 7998; Email: pchatterjee{at}nccu.edu
Received July 22, 2008. Revised September 9, 2008. Accepted September 12, 2008.
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 
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.