Building libraries of GFP-tagged neuronal proteins

描述(由申请人提供)：这是一项R21提案，旨在开发和测试一项可能对神经系统新探针的开发产生重大影响的技术，并可能在我们对基因组的了解和我们想要了解的基因之间架起一座桥梁。我们已经创造了一个Tn5转座子，它将GFP随机插入蛋白质中，产生荧光的三联融合蛋白。我们对三种蛋白质的测试表明，如果插入的方向和阅读框架正确，几乎所有转座插入编码序列的片段都会产生荧光蛋白质。此外，这些蛋白质中有六分之一继续发挥作用。我们的首要目标是改进这个工具，并对其进行更严格的测试。这将涉及用不同颜色的荧光蛋白以两种方式定位重建转座子。1)相反的方向(双头排列)将被测试以产生一种转座子，该转座子产生一种荧光蛋白，而不管它落在哪里。这些改进将使任何一次筛查中可以回收的荧光蛋白数量翻一番。如果成功，这一工具将使该领域能够更快地开发出基因可解码的活性传感器，以及优化的荧光能量转移对，可用于在活神经元中进行动力学测量。这一工具将在两对形成异寡体的蛋白质上进行测试，看看它是否能够产生荧光能量转移(FRET)对。该提议的第二个目标是以头尾相连的方式产生携带CFP和YFP的转座子。这可能是一个FRET盒式磁带，可以用来扫描蛋白质激活时向上移动的区域。这一工具将在两种蛋白质上进行测试，我们对它们的结构有很好的信息，并可以控制它们的激活。

DESCRIPTION (provided by applicant): This is an R21 proposal to develop and test a technology that may have a large impact on the development of new probes for the nervous system, and which may serve as a bridge between what we know about the genome and what we want to know about the genes it encodes. We have created a Tn5 transposon that inserts GFP randomly into the proteins, producing tribrid fusion proteins that are fluorescent. Our tests of three proteins show that virtually all of the transposed insertions into coding sequences produce fluorescent proteins if the insertion is in the correct orientation and reading frame. Moreover, 1 in 6 of these proteins continue to function. Our first aim is to improve this tool, and test it more rigorously. This will involve rebuilding the transposon with different colored fluorescent proteins positioned in two ways. 1) Opposing orientations (a double-headed arrangement) will be tested to create a transposon that produces a fluorescent protein regardless of the orientation it lands in. These improvements will double the number of fluorescent proteins that can be recovered in any one screen. If successful, this tool should empower the field to more quickly develop genetically decodable sensors of activity as well as optimized fluorescence energy transfer pairs that can be used to make kinetic measurements in living neurons. This tool will be tested on two pairs of proteins that form heteroligomers to see if it can generate fluorescence energy transfer (FRET) pairs. The second aim of the proposal is to generate a transposon carrying CFP and YFP in a head-to-tail configuration. This is potentially a FRET cassette that could be used to scan a protein for regions that move up on activation. This tool will be tested on two proteins where we have good information about their structure and can control their activation.