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

本项目的目的是建立一种体外分化心肌细胞的系统，并阐明利用该系统确定心肌细胞谱系的机制。我们构建了靶向载体，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。利用这种ES细胞来源的心肌细胞系统，我们研究了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，人们认为NKX2.5的上游一定有决定心脏细胞谱系的调控基因。我们分离了几个BAG克隆，并构建了100多个报告构建体，覆盖了NKX2.5约50kb的调控区域。我们成功地在ES细胞来源的心肌细胞中确定了6个活跃的区域。我们缩小了这6个区域(小于100kb)，并在每个调控区域的控制下产生了表达LacZ的转基因小鼠。我们目前正在分析体内这些调控区域的增强子活性。这些结果表明，ES细胞来源的心肌细胞系统是分析心脏启动子的有用工具。我们将分离和分析与NKX2.5调节区结合的调节蛋白

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