LIGHT-ACTIVATED GENE EXPRESSION IN SINGLE CELLS (RMI)

描述（由申请人提供）：该提议是开发1）一种可光活化的基因，其在暴露于光时开始转录可见的新生RNA链，和2）一种用于随后检测活细胞和组织中的单个RNA分子的方案。 我们将使用蜕皮激素反应元件和笼状的，可光活化的蜕皮激素。 我们将在基因中插入一个RNA报告基因，包含24个MS 2重复序列，因此可以通过观察与RNA转录本结合的GFP-MS 2融合蛋白来监测表达。 这些RNA已在培养的细胞中作为单分子被检测到（Fusco等人，2003年）。 我们打算将这个系统改造成癌细胞，这些癌细胞可以在培养基中生长，并可以在大鼠体内形成肿瘤。 然后，通过首先通过常规然后通过双光子显微镜在体内解开蜕皮激素来启动基因表达。 我们将使用这些细胞在体内形成肿瘤，并通过活体成像（Condeelis和Segall，2003），我们将通过显微镜使组织经受高度聚焦的光解点。 以这种方式，可以在组织内的单个细胞中观察到随后的基因表达。 单个RNA分子运动的动力学可以在活体内描述，并且在生理相关系统内检查它们的分布。 具体目标是： 1.构建具有光活化基因和mRNA报告基因的稳定细胞系。 2.光激活基因，然后真实的检测RNA转录，并对单细胞中从转录位点释放的单个RNA分子进行成像。 3.肿瘤组织内单个细胞中这种表达的活体内成像。

DESCRIPTION (provided by applicant): This proposal is to develop 1) a photoactivatable gene that, upon exposure to light, begins transcription of visible nascent chains of RNA and 2) a protocol for subsequently detecting single RNA molecules in living cells and tissues. We will use the ecdysone response element and a caged, photoactivatable ecdysone. Into the gene we will insert an RNA reporter, containing 24 MS2 repeats, so expression can be monitored by the observation of a GFP-MS2 fusion protein that binds to the RNA transcripts. These RNAs have been detected as single molecules in cultured cells (Fusco et al., 2003). We intend to engineer this system into cancer cells that can be grown in culture and can form tumors in rats. Gene expression will then be initiated by uncaging the ecdysone in vivo first by conventional and then by two-photon microscopy. We will use these cells to form tumors in vivo and by intravital imaging (Condeelis and Segall, 2003), we will subject the tissue to a highly focused spot of photolysis through the microscope. In this way, the subsequent gene expression can be observed in a single cell within a tissue. The dynamics of single RNA molecule movements could then be described intravitally, and their distributions examined within a physiologically relevant system. The Specific Aims are: 1. Constructing a stable cell line with a photoactivatable gene and an mRNA reporter. 2. Photoactivating the gene and then detecting RNA transcription in real time and imaging the single RNA molecules released from the transcription site in single cells. 3. Intravitally imaging of this expression in a single cell within tumor tissue.