Retroviral infection into myoblast
Retroviral infection into myoblast
Material
- Myoblast
- Retrovirus sol
See Retrovirus production in Plat-E cell.
- Polybrene (10 mg/ml, Millipore TR-1003-G, 4C)
- Growth medium for myoblast (Ham's/F10, 20% FBS, 0.2 ug/ml bFGF, PS)
- Differentiation medium for myoblast (DMEM/5% HS/PS)
- Puromycin (2 mg/ml stock, -20C)
Day 1
- Seed 5x10^4 myoblasts on collagen-coated 3 cm dish.
Day 2
- Change medium to 1 ml virus sol with 10 ug/ml polybrene (1/1000 of stock).
- Culture for 4 h.
- Add 1 ml growth medium.
Day 3 (24 h after infection)
- Change medium to 1.5 ml growth medium.
Day 4 (48 h after infection)
- Cells can be used as growing myoblast (day 0).
- FOR DIFFERENTIATION: Change medium to 1.5 ml differentiation medium.
- FOR PUROMYCIN SELECTION: Change medium to 1.5 ml growth medium with 2 ug/ml puromycin (1/1000 of stock).
Day 5 (24 h after puromycin selection)
- Change medium to 1.5 ml growth medium with 2 ug/ml puromycin.
Day 6 (48 h after puromycin selection)
- Cells can be used as puromycin-selected growing myoblast (day 0).
- FOR DIFFERENTIATION: Change medium to 1.5 ml differentiation medium.
