Substractive Hybridization Cloning experiments were used to find genes that were selectively expressed in cultured myoblasts, but not in undifferentiated mesoderm cells. Outline the steps you would use to do this. When MyoD is transfected into non-muscle cells in cell culture, muscle specific genes are activated. 9.9 illustrates the experimental design. Conversion of a fibroblast to a muscle cell by transfection with the MyoD1 gene.
Blue nuclei are in untransfected cells.
Spatial expression of MyoD illustrated in Northern blots from dissected tissues. MyoD1 is a protein that binds to nuclear DNA and acts as a Specific Transcription Factor. It regulates gene transcription by binding to enhancer regions in the control regions of a different muscle-specific genes. When MyoD protein is combined with an excess amount of labeled enhancer regions of the Creatine Kinase gene and the products separated by gel electrophoresis. The specificity of the interaction between MyoD and one of its target enhancers is demonstrated in the middle lane in which MyoD protein was first combined with an excess quantity of unlabeled Creatine Kinase enhancer, before adding labeled creatine kinase enhancer.