LIGHT-ACTIVATED GENE EXPRESSION IN SINGLE CELLS WITHIN TISSUE

描述（由申请人提供）：本提案的目的是获得一种与双光子显微镜兼容的光活化基因表达系统，该系统将允许在活动物组织中的单个细胞内诱导单个基因。这将提供一个有价值的资源，使两个新的研究线在基因表达：产生一个高的时间和空间分辨率的探针，用于评估转录的机制，并提供手段，以评估特定的基因产物的下游影响，在组织内的细胞，其中所有周围的细胞作为对照。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严格地作为激活基因的功能来处理。我们已经使用这种资助机制在培养细胞中为这种方法提供了原理证明。在以前的工作中，我们已经构建了一个报告基因和细胞系，可以通过蜕皮激素受体激活，从而正交哺乳动物的转录激活。在与我们的合作PI，大卫劳伦斯，合成有机化学家，我们笼蜕皮类固醇类似物ponasterone，并确定它可以被激活的激光在340 nm。我们建立了一个开放的显微镜，能够将激光束聚焦到细胞上的微米点，并证明了报告基因的激活。通过含有与来自噬菌体MS 2的荧光衣壳蛋白结合的茎环重复序列的新生RNA检测基因活化，并且该RNA被翻译成含有在过氧化物酶体中集中的序列的蓝色荧光蛋白。该提案描述了我们打算如何调整该系统，使其与活组织中的研究兼容。这涉及对双光子激发敏感的笼化组的设计和使用，使用异种移植细胞的动物模型的构建，在组织中成像的方法的发展（联合PI John Condeelis的专业知识），具有高分辨率和单分子灵敏度，并设计和操作定制的方法来收集图像（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，我们打算构建一个转基因小鼠，其中我们已经插入了一个生理相关蛋白的光激活基因，以确定其对组织中单个细胞的影响。公共卫生相关性：我们发现了一种仅使用聚焦激光束激活基因的方法。这个过程使用了一种昆虫激素，我们可以用一种可以被光分解的化合物来阻断它。我们已经建立了一个显微镜，使我们能够在活体动物的细胞和组织中这样做，这样我们就可以确切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。我们特别感兴趣的是导致癌症的蛋白质，癌基因，以及确定这些蛋白质如何诱导细胞产生肿瘤。

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.