Leiden Muscular Dystrophy pages - Protocols^©

Adenovirus-mediated MyoD gene transfer

(by Anneke Janson en Judith van Deutekom; last modified on Feb 2004)

Protocol

• fibroblast, amniocytes or chorion villi cell cultures are grown in proliferation medium
• at a confluency of 75%, cells are infected with the adenoviral vector Ad5fib50-MyoD (see Havinga et al. [2002])
  • amniocytes; 25 pfu (plaque forming units) per cell
  • chorion villi cells; 50 pfu per cell
  • fibroblasts; 75 pfu per cell
  • low myogenic myoblast cells; 75 pfu per cell
• Following a 2 h incubation at 37°C / 5% CO2, rince twice with EBBS and add differentiation medium
• The infected cell cultures are incubated at 37°C / 5% CO2 for a period of about 7-10 days, dependent on the rate of cell differentiation (i.e. myotube formation)

NOTE: initially we used a retroviral vector (see Retrovirus-mediated MyoD gene transfer)

Immunohistochemical and RNA analysis

Adenovirus-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).

Media

• proliferation medium
  • fibroblasts; DMEM supplemented with 4 mM GlutaMAX-I, 4.5 g/L glucose, 1% P/S and 10% FBS
  • amniocytes and chorion villi cells; Amniomax C100 Basal Medium supplemented with Amniomax C100 Supplement. (Invitrogen)
  • myoblasts; Nut.Mix F-10 (HAM) with Glutamax-I with 1% P/S. and 20% FBS
• infection medium; DMEM supplemented with 4 mM GlutaMAX-I, 4.5 g/L glucose, 1% P/S and 2% HS (horse serum)
• differentiation medium; DMEM supplemented with 4 mM GlutaMAX-I, 4.5 g/L glucose, 1% P/S and 2% FBS
• DMEM; Dulbecco’s Modified Eagle’s Medium (Gibco-BRL)
• FBS; (Gibco-BRL)
• P/S; penicillin/streptomycin (Gibco-BRL)
• glucose; (Gibco-BRL)
• GlutaMAX-I; (Gibco-BRL)
• HS; horse serum (Gibco-BRL)
• adenoviral MyoD-vector;  Ad5Fib50MyoD (Crucell, Leiden, Nederland)
  NOTE: avoid repeated freeze/thawing and always use virus aliquots

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