Miniaturization of cell-based β-lactamase-dependent FRET assays to ultra-high throughput formats to identify agonists of human liver X receptors
Miniaturization of cell-based β-lactamase-dependent FRET assays to ultra-high throughput formats to identify agonists of human liver X receptors
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DOI：
10.1089/154065803772613417
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发表时间：
2003-12-01
影响因子：
1.8
通讯作者：
Mitnaul, LJ
中科院分区：
文献类型：
--
作者：
Chin, J;Adams, AD;Mitnaul, LJ
肝脏X受体（LXRs）的激活诱导胆固醇逆向转运，并增加体内高密度脂蛋白胆固醇。在这里，我们描述了新的，功能性的，均相的细胞为基础的荧光共振能量转移试验，用于确定激动剂的LXRS使用β-内酰胺酶作为报告基因。产生并表征了表达LXR α-GAL 4或LXR β-GAL 4融合蛋白的稳定中国仓鼠卵巢细胞系，所述融合蛋白调节来自上游7 X UAS GAL 4 DNA结合序列的β-内酰胺酶转录。LXR的合成和天然配体以亚型特异性方式剂量依赖性地激活β-内酰胺酶的表达。这些试验用于证明I-吡啶基乙内酰脲小分子LXR合成配体特异性激活LXR α受体。β-内酰胺酶测定针对细胞密度、二甲亚砜敏感性和激动剂刺激时间进行了优化。将克隆LXR β-GAL 4-β-内酰胺酶细胞小型化为超高通量（3，456孔纳米板）筛选形式。
Activation of liver X receptors (LXRs) induces reverse cholesterol transport and increases high-density lipoprotein cholesterol in vivo. Here, we describe novel, functional, homogeneous cell-based fluorescence resonance energy transfer assays for identifying agonists of LXRS using beta-lactamase as the reporter gene. Stable Chinese hamster ovary cell lines expressing LXRalpha-GAL4 or LXRbeta-GAL4 fusion proteins that regulate beta-lactamase transcription from upstream 7 X UAS GAL4 DNA binding sequences were generated and characterized. Synthetic and natural ligands of LXR dose-dependently activated the expression of beta-lactamase in a subtype-specific manner. These assays were used to demonstrate that a I-pyridyl hydantoin small molecule LXR synthetic ligand specifically activates LXRalpha receptors. The beta-lactamase assays were optimized for cell density, dimethyl sulfoxide sensitivity, and time of agonist stimulation. Clonal LXRbeta-GAL4-beta-lactamase cells were miniaturized into an ultra high throughput (3,456-well nanoplates) screening format.