Genome-wide GFP Protein Trap

全基因组 GFP 蛋白陷阱

• 批准号: 6505381
• 负责人: GRAEME W DAVIS
• 金额: $ 17.09万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-08-15 至 2004-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6505381
• 关键词: Drosophilidae; chimeric proteins; complementary DNA; embryo /fetus; genome; green fluorescent proteins; molecular biology information system; polymerase chain reaction; protein sequence; protein structure; synapses; technology /technique development

DESCRIPTION (provided by applicant): The advent of green-fluorescent protein (GFP) as a means to visualize proteins in living cells has begun a revolution in most fields of cellular biology. Three major drawbacks currently hamper experiments using GFP-protein tagging. First, proteins of interest, once tagged, must generally be over-expressed and this over-expression can generate novel phenotypes that disrupt the processes being studied. In addition, over-expression will disrupt protein localization. Finally, to tag a protein, a full-length cDNA must be available. We have developed a GFP-protein trap in Drosophila that is capable of generating full-length GFP-fusion proteins, at random, throughout the genome. The GFP-trap is nearly random, and independent of protein size. The expression of the resulting GFP-fusion proteins will be controlled by endogenous genomic regulatory sequences. As a result, the correct spatial and temporal expression patterns will be achieved, as well as the endogenous protein expression levels. We propose develop a database of GFP-fusion proteins encompassing more than 50% of the Drosophila genome that will dramatically facilitate experiments aimed at understanding protein dynamics in living cells, in vivo. We anticipate that this technology will enable a new generation of experiments. Protein localization, trafficking, turnover, concentration and translation will be able to be studied in an in vivo genetic system without the caveat of protein over-expression that often precludes reliable experimental interpretation. Ultimately, the database that we will develop will also enable the development of new assays for cell and developmental studies based on GFP-expression in subsets of cells, determined by the expression of single genes. Our database will also facilitate the identification of new genes that function at discrete times and places involved in processes such as cell fate and neuro-development. Finally, we anticipate that this database will enable future proteomic approaches in an in vivo model genetic organism.

描述（由申请人提供）：绿色荧光蛋白（GFP）作为活细胞中蛋白质可视化手段的出现已经开始了细胞生物学大多数领域的革命。目前有三个主要的缺点阻碍了使用GFP-蛋白质标记的实验。首先，感兴趣的蛋白质一旦被标记，通常必须过表达，这种过表达可以产生新的表型，破坏正在研究的过程。此外，过度表达会破坏蛋白定位。最后，要标记蛋白质，必须有全长cDNA。我们已经在果蝇中开发了一种GFP蛋白陷阱，能够在整个基因组中随机产生全长GFP融合蛋白。GFP陷阱几乎是随机的，并且与蛋白质大小无关。所得GFP融合蛋白的表达将由内源基因组调控序列控制。因此，将实现正确的空间和时间表达模式以及内源性蛋白质表达水平。我们建议开发一个包含超过50%的果蝇基因组的GFP融合蛋白数据库，这将大大促进旨在了解活细胞中蛋白质动力学的实验。我们预计，这项技术将使新一代的实验。蛋白质的定位，运输，周转，浓度和翻译将能够在体内遗传系统中进行研究，而不需要蛋白质过度表达的警告，这通常会妨碍可靠的实验解释。最终，我们将开发的数据库还将能够开发基于细胞亚群中GFP表达的细胞和发育研究的新检测方法，这些细胞亚群由单个基因的表达决定。我们的数据库还将有助于识别在离散时间和地点发挥作用的新基因，这些基因参与细胞命运和神经发育等过程。最后，我们预计，该数据库将使未来的蛋白质组学方法在体内模型遗传生物。