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The mechanisms by which muscle gene expression is initiated and maintained are not fully understood. Muscle genes are regulated by combinatorial interactions between numerous transcription factors bound to enhancers and promoters, and their associated protein complexes. Among the most important are the MyoD and MEF2 transcription factor families, but dozens of other factors play important regulatory roles, and many additional transcription factors are certain to be involved. Expression of muscle-specific genes varies among different anatomical muscles and in fast- vs. slow-twitch fiber types, suggesting different mechanisms of regulation in response to diverse physiological cues. Thus, identifying novel transcriptional regulators and interactions is key to understanding how different cells establish the muscle phenotype; it is also critical for developing methods to combat diseases such as muscular dystrophy. Using Muscle creatine kinase as a model, we outline the key steps involved in identifying muscle gene control elements, their binding factors, and mechanisms of transcriptional activation and repression. The basic principles described here can also be applied to the transcriptional analysis of other cell-type specific genes.

Gene therapy for muscular dystrophies requires efficient gene delivery to the striated musculature and specific, high-level expression of the therapeutic gene in a physiologically diverse array of muscles. This can be achieved by the use of recombinant adeno-associated virus vectors in conjunction with muscle-specific regulatory cassettes. We have constructed several generations of regulatory cassettes based on the enhancer and promoter of the muscle creatine kinase gene, some of which include heterologous enhancers and individual elements from other muscle genes. Since the relative importance of many control elements varies among different anatomical muscles, we are aiming to tailor these cassettes for high-level expression in cardiac muscle, and in fast and slow skeletal muscles. With the achievement of efficient intravascular gene delivery to isolated limbs, selected muscle groups, and heart in large animal models, the design of cassettes optimized for activity in different muscle types is now a practical goal. In this protocol, we outline the key steps involved in the design of regulatory cassettes for optimal activity in skeletal and cardiac muscle, and testing in mature muscle fiber cultures. The basic principles described here can also be applied to engineering tissue-specific regulatory cassettes for other cell types.