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

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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