Shakes Leighcraft A, Malcolm Tennison L, Allen Kevin L, De Supriyo, Harewood Ken R, Chatterjee Pradeep K
Julius L. Chambers Biomedical/Biotechnology Research Institute, Department of Chemistry, North Carolina Central University, Durham, NC 27707, USA.
Nucleic Acids Res. 2008 Nov;36(19):6237-48. doi: 10.1093/nar/gkn628. Epub 2008 Oct 1.
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.
利用一种新方法从功能上鉴定了斑马鱼淀粉样前体蛋白基因（APPb）内含子1中的一个增强子。用增强子捕获技术改造细菌人工染色体（BAC），并作为转基因在斑马鱼中表达。瞬时分析和稳定品系的表达均用于分析。当与APPb基因启动子近端元件一起放置时，该增强子在脊索的特定非神经细胞中具有活性，但其功能局限于与该基因额外的远上游启动子元件并列时在神经元中的特异性表达，并且是这种特异性表达绝对必需的。我们证明，绿色荧光蛋白荧光的表达类似于APPb mRNA的组织分布，既需要内含子1增强子，也需要该基因上游约28 kb的DNA。结果表明，分离出的增强子的组织特异性可能与其自身基因背景下的组织特异性有很大不同。利用这种增强子和上游序列，APPb的多态变体现在可以在阿尔茨海默病动物模型中更紧密地重现APPb表达的内源性模式和调控。该方法应有助于从功能上定位BAC中有无基因编码序列的多个不连续调控元件。
