High Titer Retroviral Infections Using Virus with VSVG

(Jennifer Kennell, 2/05)

Materials needed:

• • BOSC packaging cells (Rack 5, box A in room 7615)

  • pVSVG vector (R77)

  • retroviral vector of choice (pLXSN, etc)

  • 8 mg/mL Polybrene

  • 0.45 uM syringe filters

  • syringes

• 1. Transiently transfect BOSC packaging cells with 5 µg of pVSVG and 5 µg of retroviral vector using calcium-phosphate precipitation. Shock (glycerol/PBS) and feed cells 4-6 hours after transfection.

  2. Feed transfected cells 24 hours later

  3. 48 hours after transfecting, collect media from BOSC cells and filter through 0.45 µM syringe filter. Add polybrene to final concentration of 8 µg/mL

  4. Transfer media containing virus to cells you want to infect

  5. Split cells into selection media once cells get to correct density.