Imaging Transcription in Living Animals

描述(由申请人提供)：我们申请的目标是设计和开发一种实验方法，使我们能够在活组织中实时可视化转录。这项技术将结合光学、蛋白质化学和分子生物学的专业知识。我们的方法将解决关键的技术障碍，例如，i)形成能够穿透组织并捕获其发出的荧光信号的成像光束，ii)设计一种在活组织中表达时将被标记有荧光标记的RNA，iii)选择一种发射波长在组织中透明地作用并可以被双光子吸收激发的荧光标记。我们打算可视化组织中的β-肌动蛋白基因的转录，因为它已经在所有细胞中得到了很好的表征和结构性表达。我们已经设计出转基因小鼠，在β-肌动蛋白基因的基因组拷贝中含有多个MS2干环结构。茎环对融合到荧光报告分子上的衣壳蛋白有很强的亲和力。使用双光子显微镜，我们可以观察肌肉和脑组织中的转录，而且我们的涨落分析可以告诉我们在任何给定的时间有多少聚合酶参与基因。我们收集的数据将使我们能够确定β-肌动蛋白转录的起始、延长和终止率。 在组织内的每个细胞中。由我们的方法设计的方法将有广泛的应用，并作为一种新的策略在量化组织中的基因表达水平，更重要的是推导出一般的基因表达模型，这将是理解基因在不同组织中以及同一组织内的细胞之间如何调控的基础。实时研究单个基因的动态将导致实验直接测试关于细胞分化和动态平衡过程中基因活动的随机性质的假设，并推导出一种研究疾病基因的方法。

DESCRIPTION (provided by applicant): The goal of our application is to design and develop an experimental approach that will allow us to visualize transcription in real time within living tissues. The technology will combine expertise in optics, protein chemistry and molecular biology. Our approach will address key technical hurdles such as, i) fashioning an imaging beam capable of penetrating tissue and capturing the fluorescence signal emanating from it, ii) designing an RNA that will become tagged with a fluorescent marker when expressed in living tissue, iii) selecting a fluorescent tag with an emission wavelength that acts transparently in tissue and can be excited by two-photon absorption. We intend to visualize transcription of the beta-actin gene in tissues, since it has been well-characterized and constitutively expressed in all cells. We have engineered transgenic mice that harbor multiple MS2 stem-loop structures within genomic copies of the beta-actin gene. The stem-loops have a strong affinity for a capsid protein which is fused to a fluorescent reporter. Using two-photon microscopy we can observe the transcription in muscle and brain tissue, and furthermore our fluctuation analysis can tell us how many polymerases are engaged on the gene at any given time. The data we collect will allow us to determine the initiation, elongation, and termination rates of beta-actin transcription in each cell within the tissue. The methods devised by our approach will have broad applications and serve as a novel strategy in quantifying the levels of gene expression in tissues and more importantly to derive a model for gene expression in general that will be the basis for understanding how genes are regulated in different tissues, and among cells within the same tissue. Studying dynamics of a single gene in real time will lead to experiments directly testing hypotheses concerning the stochastic nature of gene activity during cell differentiation and homeostasis as well as deriving an approach to study disease genes.