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方面的专业知识。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从中发出的荧光信号的成像光束，ii)设计一种RNA，当在活组织中表达时将被荧光标记标记，iii)选择具有发射波长的荧光标签，该荧光标签在组织中透明地起作用，并且可以被双光子吸收激发。我们打算可视化-肌动蛋白基因在组织中的转录，因为它已经被很好地表征并在所有细胞中组成性表达。我们设计了转基因小鼠，在-肌动蛋白基因的基因组拷贝中含有多个MS2茎环结构。茎环对融合荧光报告蛋白的衣壳蛋白有很强的亲和力。利用双光子显微镜，我们可以观察到肌肉和脑组织中的转录，此外，我们的波动分析可以告诉我们在任何给定时间有多少聚合酶参与到基因上。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，延伸和终止率

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.