The search for Nkx2–5-regulated genes using purified embryonic stem cell-derived cardiomyocytes with Nkx2–5 gene targeting

Cardiac transcription factors play crucial roles in cardiac development and differentiation. To address the target genes they regulate is important for understanding the molecular mechanisms underlying these processes. In this study, ES cell lines harboring a neomycin resistance gene in the Nkx2–5 gene locus were generated and used to make purified ES cell-derived cardiomyocytes (ESCM) by in vitro differentiation and successive G418 treatment. Three lines with different Nkx2–5 gene expression levels were made and named as Nkx2–5 −/−, +/−, and overexpressing ESCMs. In order to investigate Nkx2–5-regulated gene expression in cardiomyocytes, the gene expression profiles were compared among these lines. The expression patterns of known Nkx2–5 downstream genes were consistent with the previous investigations in vivo. Several genes with Nkx2–5-dependent changes in their expression were detected and confirmed by real-time PCR. This study investigated Nkx2–5-regulated gene expression in ESCM and suggested potential targets of Nkx2–5 in cardiogenesis.