FACS-based in planta BiFC screening

基于 FACS 的植物 BiFC 筛选

基本信息

批准号：
38601546
负责人：
Professor Dr. Klaus Harter
金额：
--
依托单位：
Abteilung für Pflanzenphysiologie
依托单位国家：
德国
项目类别：
Priority Programmes
财政年份：
2007
资助国家：
德国
起止时间：
2006-12-31 至 2011-12-31
项目状态：
已结题

来源：
https://gepris.dfg.de/gepris/projekt/38601546?language=en
关键词：
FACS based planta BiFC screening

FACS based planta BiFC screening

项目摘要

For the first round of the SPP1212, our proposed research was divided into two parts. In the first part we proposed the exploration of the functional role of two-component signalling systems (TCS) in Arabidopsis-microbe interactions. This part was not recommended for funding. In the second part we had proposed to establish a novel Bimolecular Fluorescence Complementation (BiFC)-based in vivo two-hybrid system for the identification and characterisation of protein-protein interactions in living plant cells by using fluorescence activated cell sorting (FACS). The establishment of the in vivo BiFC based interaction screen was more challenging than expected. However we have managed to develop a working protocol. Our approach is of great interest to most groups in and also outside of the SPP1212 that are working on in planta protein-protein interaction and protein complex formation. This proposal is targeted towards further improving the FACS-based in planta BiFC screening approach and offering the technology and expertise to the members of the SPP1212 in a dedicated fashion. Furthermore, we also offer research support, by providing dedicated cytometric and FACS expertise for other applications besides the BiFC screen to the SPP1212 community such as promoter transactivation, sorting of cells and nuclei for “-omics” work (e.g. transcriptomics, proteomics, ChIP-chip) and monitoring cell cycle (e.g. DNA content).

在SPP1212的第一轮中，我们提出的研究分为两个部分。在第一部分中，我们提出了对拟南芥微米相互作用中两个组分信号系统（TC）功能作用的探索。不建议将此部分用于资金。在第二部分中，我们提议建立一种新型的双分子荧光互补（BIFC），基于体内两杂化系统，通过使用荧光激活的细胞分类（FACS），以鉴定和表征活植物细胞中蛋白质蛋白质相互作用。基于体内BIFC的相互作用屏幕的建立比预期的要挑战。但是，我们设法开发了一个工作协议。我们在植物蛋白 - 蛋白质相互作用和蛋白质复合物形成中的SPP1212组中的大多数组都引起了极大的兴趣。该建议旨在进一步改善Planta BIFC筛选方法中的FACS，并以专门的方式向SPP1212成员提供技术和专业知识。此外，我们还通过为其他应用提供专门的细胞术和FACS专业知识，除了BIFC屏幕外，我们还提供了研究支持，例如启动子反式激活，细胞和细胞核的“ - 组学”作品的分类和细胞核的作品（例如，转录组学，蛋白质组学，CHIP-CHIP）和监视细胞周期（E.G.G.DNA contents contents contract）。