Leiden Muscular Dystrophy pages - Protocols©

MyoD-induced myo-differentiation

(Judith van Deutekom; last modified on August 13, 2001)


Purpose

Diagnosis of muscular dystrophies is often hampered by the absence of muscle tissue from the patient. We have developed the MyoD-system, an in vitro muscle differentiation system, as a way to bypass this problem. Using a retroviral or adenoviral vector the MyoD gene is delivered to patient-derived cells (fibroblasts, amniocytes or chorionic villi cells, Fig.1). Expression of the introduced MyoD gene induces differentiation of the cells into myogenic cells. Through serum deprivation, the cells further differentiate and fuse into multi-nucleated myotubes. A time-dependent range of muscle-specific proteins becomes expressed, including myosin, titin and dystrophin.

Differentiated cell cultures are harvested for RNA-isolation and mutation detection using PTT, or fixated (in cold methanol) for immunohistochemical analysis using combinations of antibodies against muscle-expressed proteins (Examples).

Although we have primarily applied the method for diagnosis of DMD/BMD, it should also be useful for diagnosis / research of other neuromuscular disorders. Currently, we are particulary interested in cell lines from patients with Limb-Girdle muscular dystrophy (LGMD).


Protocol

Since adenovirus is only moderately efficient in transducing fibroblasts when compared to amniocytes or chorion villi cells, we are still using the retroviral vector for transfer of the MyoD gene into fibroblasts


Information

Dr. Judith C.T. van Deutekom
        Department of Human and Clinical Genetics, Leiden University Medical Center
        E-mail: deutekom@lumc.nl / Tel: +31 - 71 - 527 6080

Dr. Johan T. den Dunnen
        Department of Human and Clinical Genetics, Leiden University Medical Center
        E-mail: ddunnen@lumc.nl / Tel: +31 - 71 - 527 6105



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