DEVELOPMENT OF A FLOW CYTOMETRIC YEAST TWO HYBRID SCREENING SYSTEM

该子项目是利用 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得主要资金， 因此可以在其他CRISP条目中表示。列出的机构是 中心，不一定是研究者的机构。 为了在基因组水平上发现蛋白质相互作用组图谱，基于酵母双杂交的筛选系统在过去的十年中得到了广泛的应用。 尽管在适应液体处理机器人和阵列仪器方面做出了巨大努力，但由于基于琼脂平板的营养标记选择和LacZ报告基因分析，目前的Y2 H作图方法仍然是劳动密集型和低通量的。 我们最近开发的基于yEGFP的酵母双杂交系统使得能够通过流式细胞术定量分析Y2 H测定培养物的GFP荧光而无需铺板。 随着HyperCyt <$高通量流式细胞仪采样平台的最新仪器进步，可以在不到20分钟的时间内定量分析384个Y2 H液体培养样品的平板。 虽然这样的系统非常适合于分析二元蛋白质-蛋白质相互作用对，但它不具有足够的通量来进行大的cDNA文库筛选，例如分选大的人cDNA文库细胞（通常约109个细胞）可能需要数天。 为了解决这一限制，我们正在建立一种新的sdY 2 H系统，该系统将表面展示报告基因作为额外的选择标记，允许通过简单的磁性分离步骤快速富集阳性细胞群。 这种新的表面报告基因与先前建立的yEGFP报告基因系统一起，能够在液体培养中快速筛选cDNA文库，为传统的基于平板的筛选和分析提供了高通量的替代方案。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. To discover protein interactome maps at genomic scales, yeast two hybrid-based screening system has been used extensively in the past decade. Despite the tremendous effort in adapting liquid handling robot and array instrumentation, the current Y2H mapping approach is still labor intensive and low throughput attributed to the agar plating-based nutrient marker selection and LacZ reporter analysis. Our recent development of a yEGFP-based yeast two hybrid system enabled the quantitative analysis of GFP fluorescence of the Y2H assay cultures by flow cytometry without plating. With the recent instrumentation advancement of the HyperCyt¿ high-throughput flow cytometry sampling platform, a plate of 384 Y2H liquid culture samples can be quantitatively analyzed in less than 20 minutes. While such system is well-suited for analyzing binary protein-protein interaction pairs, it does not have adequate throughput to perform large cDNA library screening, e.g. sorting a large human cDNA library cells (typically ~109 cells) could take days. To address this limitation, we are establishing a novel sdY2H system that incorporates the surface display reporter as an additional selection marker allowing rapid enrichment of positive cell populations via a simple magnetic separation step. This new surface reporter together with the previously established yEGFP reporter system enable rapid cDNA library screening in liquid cultures, providing a high throughput alternative to conventional plating-based screening and analysis.