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Retrovirus-mediated MyoD gene transfer

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

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

• fibroblasts are grown in proliferation medium
• at a confluency of 75%, cells are infected with the retroviral vector (pLMDSN; Weintraub et al., 1989) in infection medium using a MOI of approximately 10 cfu (colony forming units) per cell
• following a 48 hr incubation at 37°C / 5% CO₂, the infection medium is replaced by selection medium
• upon selection of transduced cells for 3 days, the cells are grown to a confluency of 90%
• cell cultures are then subsequently incubated in differentiation medium for a period of about 14-20 days, dependent on the rate of cell differentiation (i.e. myotube formation)

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).

Media

• proliferation medium; DMEM supplemented with 4 mM GlutaMAX-I, 4.5 g/L glucose and 10% FBS
• infection medium; DMEM supplemented with 4 mM GlutaMAX-I, 4.5 g/L glucose, 10% FBS and 4 mg/ml polybrene
• selection medium; DMEM supplemented with 4 mM GlutaMAX-I, 4.5 g/L glucose, 10% FBS and 200-400 µg/ml G418
• differentiation medium; DMEM supplemented with 4 mM GlutaMAX-I, 4.5 g/L glucose and 2% FBS
  
• DMEM; Dulbecco’s Modified Eagle’s Medium (Gibco-BRL)
• FBS; .... (Gibco-BRL)
• G418; (Promega)
• glucose; (Gibco-BRL)
• GlutaMAX-I; (Gibco-BRL)
• polybrene; ....

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