LIGHT-ACTIVATED GENE EXPRESSION IN SINGLE CELLS(RMI)

描述(申请人提供)：这项提议是为了开发1)一种光激活基因，当暴露在光下时，它开始转录可见的新生RNA链，以及2)一种随后检测活细胞和组织中单个RNA分子的方案。我们将使用蜕皮激素反应元件和笼子里的、可光激活的蜕皮激素。我们将在基因中插入一个包含24个MS2重复的RNA报告程序，因此可以通过观察与RNA转录物结合的GFP-MS2融合蛋白来监测其表达。这些RNA已在培养细胞中被检测到为单分子(Fusco等人，2003年)。我们打算将这个系统改造成可以在培养中生长并在大鼠身上形成肿瘤的癌细胞。然后，基因表达将通过在体内去除蜕皮激素来启动，首先是常规的，然后是双光子显微镜。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)，我们将通过显微镜将组织置于高度聚焦的光解斑点。通过这种方式，可以在组织内的单个细胞中观察到后续的基因表达。然后，单个RNA分子运动的动力学可以在生命内描述，它们的分布可以在一个生理相关的系统中进行检查。 具体目标是： 1.构建具有光激活基因和信使核糖核酸报告基因的稳定细胞系。 2.光激活基因，实时检测RNA转录，并对单个细胞从转录位点释放的单个RNA分子进行成像。 3.在肿瘤组织内的单个细胞内进行这种表达的活体成像。

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.