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Establishment of ES cell-derived cardiac myocyte culture system and clarification of the mechanism for determination of the cardiac cell lineage

ES细胞来源的心肌细胞培养体系的建立及心肌细胞谱系测定机制的阐明

基本信息

批准号：
13832003
负责人：
TANAKA Makoto
金额：
$ 2.69万
依托单位：
Kyoto University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2001
资助国家：
日本
起止时间：
2001 至 2002
项目状态：
已结题

项目摘要

The aim of this project was to establish an in vitro system of differentiating cardiac myocytes and to clarify the mechanism for the determination of the cardiac cell lineage using this system. We constructed a targeting vector and replaced the first exon of the MLC2V gene with the GFP gene by homologous recombination in ES cells. Using this ES cell-derived cardiac myocyte system, we have investigated the mechanism for the determination of the cardiac cell lineage. Nkx2.5 plays a critical role at the early stage of cardiac development and it is thought that there must be regulatory genes upstream of Nkx2.5 that determine the cardiac cell lineage. We isolated several BAG clones and made more than 100 reporter constructs that covered approximately 50kb of the regulatory regions of Nkx2.5. We successfully identified 6 regions that were active in the ES cell-derived cardiac myocytes. We narrowed down these 6 regions (less than 100kb) and generated transgenic mice expressing LacZ under the control of each of these regulatory regions. We are currently analyzing enhancer activities of these regulatory regions in vivo. These results indicated that the ES cell-derived cardiac myocyte system is a useful tool for cardiac promoter analysis. We are going to isolate and analyze regulatory proteins that bind to the regulatory region of Nkx2.5

本课题的目的是建立一个体外心肌细胞分化系统，并阐明利用该系统进行心肌细胞谱系鉴定的机制。我们构建了一个靶向载体，并在ES细胞中通过同源重组将MLC2V基因的第一个外显子替换为GFP基因。使用这种ES细胞衍生的心肌细胞系统，我们已经研究了用于确定心肌细胞谱系的机制。Nkx2.5在心脏发育的早期阶段起着关键作用，并且认为Nkx2.5上游必须存在决定心脏细胞谱系的调控基因。我们分离了几个BAG克隆，并制作了超过100个报告构建体，覆盖了Nkx2.5的约50kb的调控区。我们成功地鉴定了ES细胞衍生的心肌细胞中的6个活性区域。我们缩小了这6个区域（小于100kb），并产生了在这些调控区域的控制下表达LacZ的转基因小鼠。我们目前正在分析这些调节区域的增强子活性。这些结果表明，ES细胞来源的心肌细胞系统是一个有用的工具，心脏启动子分析。我们将分离并分析与Nkx2.5调控区结合的调控蛋白。