Banting and Best Department of Medical Research, The Terrence Donnelly Centre for Cellular and Biomolecular Research (CCBR), University of Toronto, Toronto, Ontario, Canada.
PLoS One. 2010 Mar 22;5(3):e9797. doi: 10.1371/journal.pone.0009797.
Nuclear receptors (NRs) belong to a superfamily of transcription factors that regulate numerous homeostatic, metabolic and reproductive processes. Taken together with their modulation by small lipophilic molecules, they also represent an important and successful class of drug targets. Although many NRs have been targeted successfully, the majority have not, and one third are still orphans. Here we report the development of an in vivo GFP-based reporter system suitable for monitoring NR activities in all cells and tissues using live zebrafish (Danio rerio). The human NR fusion proteins used also contain a new affinity tag cassette allowing the purification of receptors with bound molecules from responsive tissues. We show that these constructs 1) respond as expected to endogenous zebrafish hormones and cofactors, 2) facilitate efficient receptor and cofactor purification, 3) respond robustly to NR hormones and drugs and 4) yield readily quantifiable signals. Transgenic lines representing the majority of human NRs have been established and are available for the investigation of tissue- and isoform-specific ligands and cofactors.
核受体（NRs）属于转录因子的超家族，可调节许多体内平衡、代谢和生殖过程。与它们被小分子脂溶性物质调节相结合，它们也代表了一类重要且成功的药物靶点。尽管许多 NRs 已被成功靶向，但大多数并未被靶向，三分之一仍为孤儿受体。在这里，我们报告了一种基于 GFP 的体内报告系统的开发，该系统适用于使用活体斑马鱼（Danio rerio）监测所有细胞和组织中的 NR 活性。所使用的人 NR 融合蛋白还包含一个新的亲和标签盒，允许从响应组织中纯化与结合分子结合的受体。我们表明，这些构建体 1）如预期的那样对内源性斑马鱼激素和辅助因子作出反应，2）促进受体和辅助因子的有效纯化，3）对 NR 激素和药物作出强烈反应，4）产生易于定量的信号。已建立了代表大多数人 NR 的转基因系，可用于研究组织和亚型特异性配体和辅助因子。
