Leiden Muscular Dystrophy pages - Protocols©

Retrovirus-mediated MyoD gene transfer

(by Judith van Deutekom; last modified on March 25, 2000)


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

Immunohistochemical and RNA analysis

Adenovirus- or retrovirus-infected 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 desmin (expressed at early myogenesis), embryonic myosin and dystrophin (both expressed at later stages of myogenesis), (double-)stainings are performed to determine the transduction efficiencies, the rate of cell differentiation and the absence or presence of dystrophin (see Examples).


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