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: 7694283
• 负责人: Robert H Singer
• 金额: $ 71.81万
• 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/7694283
• 关键词: Address; Affect; Animal Model; Animals; Binding; Breast Carcinoma; Cancer Etiology; Capsid Proteins; Cell Line; Cells; Cleaved cell; Collaborations; Computer Analysis; Coupled; Cultured Cells; Detection; Ecdysteroids; Enterobacteria phage MS2; 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; 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; public health relevance; 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（由申请人提供）：该提案的目的是推导与两光子显微镜兼容的可相关基因表达系统，该系统将允许在活动物组织中的单个细胞中诱导单个基因。这将提供一个有价值的资源，可实现基因表达中的两种新的研究线：生成高的时间和空间分辨率探测器，以评估转录机制，并提供评估所有周围细胞中组织中特定基因产物在组织中特定基因产物的下游效应的手段。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介绍。我们已经使用此资金机制为这种方法提供了这种方法的原理。在此前的工作中，我们构建了一个记者基因和细胞系，该基因和细胞系可以通过ecdysteroid受体激活，因此与哺乳动物的转录激活正交。与我们的Co-Pi合作，合成有机化学家David Lawrence合作，我们笼罩了ecdysteroid Analog Ponasterone，并确定它可以在340nm处被激光激活。我们构建了一个分离显微镜，该显微镜能够将激光束聚焦于细胞上的微米，并证明了报告基因的激活。通过含有茎环重复的新生RNA检测到该基因激活，该重复结合了噬菌体MS2的荧光衣壳蛋白，该RNA被转化为蓝色荧光蛋白，其中含有过氧化物酶体中浓缩的序列。该提案描述了我们打算如何适应该系统，以使其与活组织的研究兼容。 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）。最后，我们打算构造一种转基因小鼠，在该小鼠中插入了一个可与生理相关蛋白的可相关基因，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重点激光束激活基因的方法。该过程使用一种昆虫激素，我们可以用它可以用光裂解的化合物阻塞。我们已经建立了一个显微镜，使我们能够在活动物的细胞和组织中进行此操作，以便我们可以准确研究基因的打开方式。这使我们能够诱导单个细胞合成任何蛋白质，然后确定该蛋白在细胞中的作用。这可以控制细胞中其他蛋白质的方法和地点。我们对引起癌症，癌基因的蛋白质特别感兴趣，并确定这些蛋白质如何诱导细胞产生肿瘤。