LIGHT-ACTIVATED GENE EXPRESSION IN SINGLE CELLS

描述（由申请人提供）：该提案旨在开发1)一种光激活基因，该基因在暴露于光下时开始转录可见的新生RNA链，以及2)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型光激活蜕皮激素。我们将在基因中插入一个包含24个MS2重复序列的RNA报告基因，这样就可以通过观察GFP-MS2融合蛋白与RNA转录物的结合来监测其表达。这些rna在培养细胞中被检测为单分子（Fusco et al., 2003）。我们打算把这个系统改造成癌细胞，在培养物中生长，并在大鼠体内形成肿瘤。基因表达将首先通过常规的双光子显微镜在体内释放蜕皮激素，然后通过双光子显微镜启动。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点上。通过这种方法，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体内描述，并在生理相关系统中检查其分布。

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.