LIGHT-ACTIVATED GENE EXPRESSION IN SINGLE CELLS WITHIN TISSUE

描述（由申请人提供）：本提案的目的是推导一种与双光子显微镜兼容的光激活基因表达系统，该系统将允许在活体动物组织中的单个细胞内诱导单个基因。这将为两个新的基因表达研究提供宝贵的资源：产生一个高时间和空间分辨率的探针来评估转录机制，并提供方法来评估组织内所有周围细胞作为对照的细胞中特定基因产物的下游效应。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功能严格处理。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一个原理证明。在之前的工作中，我们构建了一个可以通过表皮甾体受体激活的报告基因和细胞系，因此与哺乳动物的转录激活是正交的。在与我们的共同负责人，合成有机化学家David Lawrence的合作下，我们将类似于表皮甾体的ponasterone放入笼中，并确定它可以被340nm的激光激活。我们建立了一个非封闭显微镜，它能够将激光束聚焦到细胞上的一个微米点上，并展示了报告基因的激活。基因激活是通过新生RNA检测到的，这些新生RNA含有茎环重复序列，该重复序列与噬菌体MS2的荧光衣壳蛋白结合，该RNA被翻译成蓝色荧光蛋白，其中包含一个集中在过氧化物酶体中的序列。该提案描述了我们打算如何调整该系统，使其与活组织的研究兼容。这包括设计和使用对双光子激发敏感的笼子组，使用异种移植细胞构建动物模型，开发具有高分辨率和单分子灵敏度的组织成像方法（联合pi John Condeelis的专业知识），设计和操作定制的方法来收集图像（联合pi Ben Ovryn的专业知识）和强大的计算分析工具（联合pi Lin Ji的专业知识）。最后，我们打算构建一个转基因小鼠，我们在其中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：我们已经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切割掉的化合物来阻止它。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开启的。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制造方式和位置。我们对导致癌症的蛋白质，致癌基因，以及确定这些蛋白质如何诱导细胞产生肿瘤特别感兴趣。

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.