Transgenic Reporters for Cardiac Growth and Regeneration

描述（由申请人提供）：心肌细胞细胞周期活性和心肌组织再生的记录通常需要大量的组织化学处理，并且经常依赖于使用主观标准进行细胞谱系测定。虽然报告转基因可以极大地帮助这样的分析，它们的使用也需要相当大的组织处理，目前的转基因报告系统不容易允许监测累积的再生生长。在该R21申请中提出的研究将产生转基因报告模型，其可用于以最小的组织处理定量心肌细胞细胞周期活性和累积心肌再生。目的1将利用心肌细胞限制性启动子来靶向表达具有内在荧光活性的蛋白质与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。心肌细胞的细胞周期状态可以简单地通过监测细胞核内报告蛋白的荧光模式来定量。目的二是建立一个三级转基因报告系统，用于定量成人心肌的累积新生生长。该系统将利用现有的条件性Cre重组酶转基因模型，结合两个新的条件性转基因，在所有经历从头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，由心肌细胞增殖引起的累积心肌生长可以简单地通过对具有核EGFP荧光的细胞的数量进行评分来确定。我们将利用现有的转基因模型，表现出增强心肌细胞增殖，以验证报告基因系统。一旦得到验证，我们将开发自动数据采集和分析协议。本申请中提出的报告转基因将具有优于现有模型的明显优势，因为可以用最少的样品处理（本质上，仅需要组织固定和切片）获得数据。此外，该系统将允许量化累积的再生增长，这不能很容易地确定与现有的模型。使用自动化技术进行数据采集和分析，应允许精确定量的低频事件，并使用荧光报告将允许活组织中的分析。

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.