Whitney M, Rockenstein E, Cantin G, Knapp T, Zlokarnik G, Sanders P, Durick K, Craig F F, Negulescu P A
Aurora Biosciences Corporation, San Diego, CA 92121, USA.
Nat Biotechnol. 1998 Dec;16(13):1329-33. doi: 10.1038/4302.
We describe a genome-wide functional assay for rapid isolation of cell clones and genetic elements responsive to specific stimuli. A promoterless beta-lactamase reporter gene was transfected into a human T-cell line to generate a living library of reporter-tagged clones. When loaded with a cell-permeable fluorogenic substrate, the cell library simultaneously reports the expression of a large number of endogenous genes. Flow cytometry was used to recover individual clones whose reporter-tagged genes were either induced or repressed following T-cell activation. Responsive clones were expanded and analyzed pharmacologically to identify patterns of regulation associated with specific genes. Although demonstrated using T cells, the genomic assay could be applied to map downstream transcriptional consequences for any propagating cell line in response to any stimulus of interest.
我们描述了一种全基因组功能分析方法，用于快速分离对特定刺激有反应的细胞克隆和遗传元件。将无启动子的β-内酰胺酶报告基因转染到人T细胞系中，以生成报告基因标记克隆的活细胞文库。当加载细胞可渗透的荧光底物时，细胞文库可同时报告大量内源性基因的表达。利用流式细胞术回收单个克隆，其报告基因标记的基因在T细胞激活后被诱导或抑制。对有反应的克隆进行扩增并进行药理学分析，以确定与特定基因相关的调控模式。尽管该基因组分析方法是在T细胞上进行验证的，但它可应用于绘制任何增殖细胞系对任何感兴趣刺激的下游转录结果。
