Imaging Transcription in Living Animals

活体动物的成像转录

基本信息

批准号：
8604155
负责人：
Robert H Singer
金额：
$ 59.1万
依托单位：
ALBERT EINSTEIN COLLEGE OF MEDICINE
依托单位国家：
美国
项目类别：
财政年份：
2012
资助国家：
美国
起止时间：
2012-04-01 至 2016-01-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8604155
关键词：
Actins Acute Address Affinity Animal Model Animals Binding Biological Sciences Biophysics Brain Capsid Proteins Cell Differentiation process Cells Characteristics Cultured Cells DNA Data Detection Development Developmental Process Disease Engineering Environment Event Faculty Fluorescence Fluorescence Microscopy Foundations Gene Expression Genes Genetic Transcription Genomics Goals Homeostasis Image Image Analysis Individual Label Lead Life Messenger RNA Methods Microscopy Modeling Molecular Molecular Biology Mus Muscle Natural regeneration Nature Optics Organ Polymerase Process Protein Chemistry Protein Engineering Proteins RNA Regulation Reporter Research Resolution Signal Transduction Site Slice Structure Technology Testing Time Tissues Training Transcription Initiation Transgenic Animals Transgenic Mice Work absorption animal tissue base beta Actin brain tissue design imaging probe improved in vivo infrared microscopy innovation member mouse model novel novel strategies physical science research study signal processing simulation stem two-photon

项目摘要

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.

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内实时可视化转录。该技术将结合光学、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其发出的荧光信号的成像光束，ii）设计一种在活体组织中表达时将被荧光标记标记的RNA，iii）选择发射波长在组织中透明作用并可通过双光子吸收激发的荧光标签。我们打算可视化组织中β-肌动蛋白基因的转录，因为它已被充分表征并在所有细胞中组成型表达。我们已经改造了转基因小鼠，在 β-肌动蛋白基因的基因组拷贝中包含多个 MS2 茎环结构。茎环对与荧光报告基因融合的衣壳蛋白具有很强的亲和力。使用双光子显微镜，我们可以观察肌肉和脑组织中的转录，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该基因。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、延伸和终止率在组织内的每个细胞中。我们的方法设计的方法将具有广泛的应用，并作为量化组织中基因表达水平的新策略，更重要的是导出一般基因表达的模型，这将成为理解基因在不同组织中如何调节的基础。组织之间，以及同一组织内的细胞之间。实时研究单个基因的动态将导致直接测试有关细胞分化和稳态过程中基因活性的随机性质的假设的实验，并得出研究疾病基因的方法。