Transgenic Reporters for Cardiac Growth and Regeneration

描述（由申请人提供）：心肌细胞周期活动和心肌组织再生的记录通常需要广泛的组织化学处理，并且经常依赖于使用主观标准来确定细胞谱系。虽然报告基因可以极大地辅助这种分析，但它们的使用也需要大量的组织处理，而且目前的转基因报告系统还不容易监测累积的再生生长。本R21申请中提出的研究将产生转基因报告细胞模型，该模型可用于量化心肌细胞周期活性和累积心肌再生，仅需最少的组织处理。目的1将利用心肌细胞限制性启动子靶向具有内在荧光活性的蛋白质和在亚核定位中经历细胞周期依赖性变化的蛋白质之间的融合表达。心肌细胞周期状态可以简单地通过监测报告蛋白荧光在细胞核内的模式定量。目的2将开发一个三级转基因报告系统来量化成人心脏的累积新生心肌生长。该系统将利用现有的条件cre -重组酶转基因模型，结合两种新的条件转基因，在所有经历新生增殖的成年心肌细胞中永久激活核定位的EGFP报告蛋白。因此，心肌细胞增殖引起的累积心肌生长可以简单地通过记录细胞核EGFP荧光细胞的数量来确定。我们将利用现有的转基因模型，显示增强心肌细胞增殖来验证这两个报告基因系统。一旦验证，我们将开发自动数据采集和分析协议。在本应用中提出的报告基因将比现有模型具有明显的优势，因为数据可以通过最少的样本处理获得（本质上，只需要组织固定和切片）。此外，该系统将允许对累积再生生长进行量化，这是现有模型无法轻易确定的。使用自动化技术进行数据采集和分析，应该可以对低频事件进行精确定量，荧光报告器的使用将允许对活组织进行分析。

DESCRIPTION (provided by applicant): Documentation of cardiomyocyte cell cycle activity and myocardial tissue regeneration typically requires extensive histochemical processing and frequently relies on the use of subjective criteria for cell lineage determination. Although reporter transgenes can greatly assist such analyses, their use also requires considerable tissue processing, and current transgenic reporter systems do not readily permit monitoring of cumulative regenerative growth. The studies proposed in this R21 application will generate transgenic reporter models which can be used to quantitate cardiomyocyte cell cycle activity and cumulative myocardial regeneration with minimal tissue processing. Aim 1 will utilize a cardiomyocyte-restricted promoter to target expression of a fusion between proteins with intrinsic fluorescent activity and proteins which undergo cell cycle- dependent changes in sub-nuclear localization. Cardiomyocyte cell cycle status can be quantitated simply by monitoring the pattern of reporter protein epifluorescence within the nucleus. Aim 2 will develop a tertiary transgenic reporter system to quantitate cumulative de novo myocardial growth in adult hearts. The system will utilize an existing conditional Cre-recombinase transgenic model, in combination with two new conditional transgenes, to permanently activate a nuclear localized EGFP reporter protein in all adult cardiomyocytes undergoing de novo proliferation. Consequently, cumulative myocardial growth resulting from cardiomyocyte proliferation can be determined simply by scoring the number of cells with nuclear EGFP epifluorescence. We will utilize existing transgenic models that exhibit enhanced cardiomyocyte proliferation to validate both reporter gene systems. Once validated, we will develop automated data acquisition and analyses protocols. The reporter transgenes proposed in this application will have the distinct advantage over existing models in that data can be acquired with minimal sample processing (in essence, requiring only tissue fixation and sectioning). Moreover, the systems will permit quantitation of cumulative regenerative growth, which cannot easily be determined with existing models. The use of automated techniques for data acquisition and analysis should permit precise quantitation of low-frequency events, and the use of fluorescent reporters will permit analyses in living tissue.