BMP induction of cardiogenesis in P19 cells requires prior cell-cell interaction(s).

Mouse P19 embryonal carcinoma cells undergo cardiogenesis in response to high density and DMSO. We have derived a clonal subline that undergoes cardiogenesis in response to high density, but without requiring exposure to DMSO. The new subline retains the capacity to differentiate into skeletal muscle and neuronal cells in response to DMSO and retinoic acid.

Differences in the function of three conserved E-boxes of the muscle creatine kinase gene in cultured myocytes and in transgenic mouse skeletal and cardiac muscle.

The 1256-base pair enhancer-promoter of the mouse muscle creatine kinase gene includes three CAnnTG E-boxes that are conserved among mammals and have flanking and middle sequences conforming to consensus muscle regulatory factor binding sites. This study seeks to determine whether these E-boxes are critical for muscle creatine kinase expression in physiologically distinct muscles. Mutations of the "right" and "left" E-boxes in the enhancer decreased expression in cultured skeletal myocytes approximately 10- and 2-fold, respectively, whereas a "promoter" E-box mutation had little effect.

Transgenic and tissue culture analyses of the muscle creatine kinase enhancer Trex control element in skeletal and cardiac muscle indicate differences in gene expression between muscle types.

The muscle creatine kinase (MCK) gene is expressed at high levels only in differentiated skeletal and cardiac muscle. The activity of the cloned enhancer-promoter has previously been shown to be dependent on the Trex element which is specifically bound by a yet unidentified nuclear factor, TrexBF. We have further characterized the function of the Trex site by comparing wild-type and Trex-mutated MCK transgenes in five mouse skeletal muscles: quadriceps, extensor digitorum longus (EDL), soleus, diaphragm, and distal tongue, as well as in heart ventricular muscle.

Analysis of muscle creatine kinase regulatory elements in recombinant adenoviral vectors.

Adenoviral gene transfer holds promise for gene therapy, but effective transduction of a large and distributed tissue such as muscle will almost certainly require systemic delivery. In this context, the use of muscle-specific regulatory elements such as the muscle creatine kinase (MCK) promoter and enhancer will avoid potentially harmful ectopic expression of transgenes. We describe here the development and testing of adenoviral vectors containing small, striated muscle-specific, highly active MCK expression cassettes.

Analysis of muscle creatine kinase gene regulatory elements in skeletal and cardiac muscles of transgenic mice.

Regulatory regions of the mouse muscle creatine kinase (MCK) gene, previously discovered by analysis in cultured muscle cells, were analyzed in transgenic mice. The 206-bp MCK enhancer at nt-1256 was required for high-level expression of MCK-chloramphenicol acetyltransferase fusion genes in skeletal and cardiac muscle; however, unlike its behavior in cell culture, inclusion of the 1-kb region of DNA between the enhancer and the basal promoter produced a 100-fold increase in skeletal muscle activity. Analysis of enhancer control elements also indicated major differences between their properties in transgenic muscles and in cultured muscle cells.