LIGHT-ACTIVATED GENE EXPRESSION IN SINGLE CELLS WITHIN TISSUE

组织内单细胞中的光激活基因表达

• 批准号: 7556680
• 负责人: Robert H Singer
• 金额: $ 71.17万
• 依托单位: ALBERT EINSTEIN COLLEGE OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-09-30 至 2012-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7556680
• 关键词: Address; Affect; Animal Model; Animals; Binding; Breast Carcinoma; Cancer Etiology; Capsid Proteins; Cell Line; Cells; Cleaved cell; Collaborations; Compatible; Computer Analysis; Coupled; Cultured Cells; Detection; Ecdysteroids; Enterobacteria phage MS2; Facility Construction Funding Category; Funding Mechanisms; Gene Activation; Gene Expression; Genes; Genetic Transcription; Image; Insect Hormones; Investigation; Lasers; Life; Light; Malignant Epithelial Cell; Mammary Neoplasms; Methods; Microscope; Microscopy; Modeling; Oncogenes; Operative Surgical Procedures; Procedures; Proteins; Public Health; Purpose; RNA; Reporter Genes; Research; Resolution; Resources; Spottings; System; Testing; Time; TimeLine; Tissues; Transcriptional Activation; Transgenic Mice; Translating; Work; Xenograft procedure; analog; design; ecdysteroid receptor; interest; method development; neoplastic cell; novel; peroxisome; single molecule; stem; tool; tumor; two-photon

DESCRIPTION (provided by applicant): The purpose of this proposal is to derive a photo-activatable gene expression system compatible with two-photon microscopy that will allow the induction of a single gene within a single cell in the tissue of a living animal. This will provide a valuable resource enabling two novel lines of research in gene expression: generating a high temporal and spatial resolution probe for assessing the mechanism of transcription and affording the means to evaluate the downstream effects of a particular gene product in a cell within tissue where all the surrounding cells serve as controls. Hence the microenvironment of the affected cell and its interactions with neighboring cells can be rigorously addressed as a function of the activated gene. We have provided a proof-of- principle for this approach in cultured cells using this funding mechanism. In this previous work, we have constructed a reporter gene and cell line that can be activated through the ecdysteroid receptor and is thereby orthogonal to mammalian transcriptional activation. In collaboration with our co-PI, David Lawrence, a synthetic organic chemist, we caged the ecdysteroid analog ponasterone and established that it could be activated by a laser at 340nm. We built an uncaging microscope that was capable of focusing the laser beam into a micron spot on a cell and demonstrated the activation of a reporter gene. The gene activation was detected through nascent RNAs containing a stem-loop repeat that binds a fluorescent capsid protein from the phage MS2 and this RNA, is translated into a blue fluorescent protein containing a sequence that concentrates in peroxisomes. This proposal describes how we intend to adapt this system so that it is compatible with investigations in living tissues. This involves the design and use of a caging group that is sensitive to two-photon excitation, the construction of animal models using xenograft cells, the development of methods to image into tissues (the expertise of the Co-PI John Condeelis) with high resolution and single molecule sensitivity and the design and operation of customized approaches to collect the images (the expertise of the Co-PI Ben Ovryn) and powerful computational analysis tools (the expertise of the Co-PI Lin Ji). Finally, we intend to construct a transgenic mouse into which we have inserted a photo-activatable gene for a physiologically relevant protein, to determine its effects on a single cell in a tissue. Public Health Relevance: We have discovered a way to activate a gene using only a focused laser beam. The procedure uses an insect hormone that we can block with a compound that can be cleaved off by light. We have built a microscope that allows us to do this in cells and tissues in a living animal so we can investigate exactly how genes turn on. This enables us to induce a single cell to synthesize any protein and then determine the actions of that protein in cells. that controls how and where other proteins are made in the cell. We are particularly interested in proteins that cause cancer, oncogenes, and in determining how these proteins can induce cells to produce tumors.

描述(申请人提供)：这项提议的目的是获得一种与双光子显微镜兼容的可光激活的基因表达系统，该系统将允许在活动物组织的单个细胞内诱导单个基因。这将为基因表达的两条新的研究路线提供宝贵的资源：产生一个用于评估转录机制的高时间和空间分辨率的探针，并提供一种手段来评估特定基因产物在组织内细胞中的下游影响，其中所有周围细胞作为对照。因此，受影响细胞的微环境及其与邻近细胞的相互作用可以作为激活基因的函数严格处理。我们已经在使用这种资助机制的培养细胞中为这种方法提供了原则上的证明。在之前的工作中，我们构建了一个报告基因和细胞系，它可以通过蜕皮类固醇受体激活，从而与哺乳动物的转录激活正交。在我们的合作伙伴，合成有机化学家大卫·劳伦斯的合作下，我们将蜕皮激素类似物重方石关在笼子里，并确定它可以被340 nm的激光激活。我们建造了一台非老化显微镜，能够将激光聚焦到细胞上的微米级斑点，并展示了报告基因的激活。通过含有茎环重复的新生RNA检测到基因的激活，该RNA与来自噬菌体MS2的荧光衣壳蛋白结合，该RNA被翻译成含有集中在过氧体中的序列的蓝色荧光蛋白。这份提案描述了我们打算如何调整这一系统，使其与活体组织的研究相兼容。这包括设计和使用对双光子激发敏感的笼组，使用异种细胞构建动物模型，开发高分辨率和单分子灵敏度的组织成像方法(Co-Pi John Condeelis的专业知识)，设计和操作定制的图像收集方法(Co-Pi Ben Ovryn的专业知识)和强大的计算分析工具(Co-Pi Lin Ji的专业知识)。最后，我们打算构建一只转基因小鼠，我们在其中插入了一种生理相关蛋白质的光激活基因，以确定它对组织中单个细胞的影响。与公共健康相关：我们已经发现了一种仅使用聚焦激光就能激活基因的方法。这个过程使用了一种昆虫荷尔蒙，我们可以用一种可以被光分解的化合物来阻止这种荷尔蒙。我们已经建造了一种显微镜，可以让我们在活着的动物的细胞和组织中做到这一点，这样我们就可以准确地研究基因是如何启动的。这使我们能够诱导单个细胞合成任何蛋白质，然后确定该蛋白质在细胞中的作用。它控制着细胞中其他蛋白质的合成方式和位置。我们对致癌蛋白质和致癌基因特别感兴趣，并确定这些蛋白质如何诱导细胞产生肿瘤。