APPLICATION OF IN-VITRO MYO-DIFFERENTIATION OF NONMUSCLE CELLS TO ENHANCE GENE-EXPRESSION AND FACILITATE ANALYSIS OF MUSCLE PROTEINS

Introduction of the myogenic-determination gene MyoD forces non-muscle cell cultures into myogenesis, thereby inducing expression of muscle- specific proteins and facilitating their analysis. In several MyoD-tra nsfected fibroblasts, immunohistochemical detection showed expression of desmin after three days, of titin after five days and of dystrophin after seven days. Cell fusion (myotube formation) could be observed a fter five days. After nine days a fraction of the cells showed a stria ted titin pattern, indicating an advanced state of muscle differentiat ion. Dystrophin (the protein absent in Ducbenne Muscular Dystrophy pat ients) can be detected in MyoD-transfected and differentiated fibrobla sts from healthy individuals, and is absent in those of patients. MyoD -transfection increases transcription of the dystrophin gene, facilita ting RNA-based mutation detection. Using RNA from MyoD-transfected, di fferentiated fibroblasts of a deceased patient with an unknown, non-de letion mutation, we were able to identify a CGA-->TGA nonsense mutatio n in the rod domain at basepair 6492 and to establish a rapid mutation specific test for future diagnosis of the mutation in his relatives. Copyright (C) 1996 Elsevier Science Ltd.