Protocol for Production of Lentiviral Vectors in 293T cells

 

Day 1 Plating (9-10am)

Plate 2-2.5x10⁶ of 293T cells per 10cm plate

Day 2 Transfection (9-10am)

Prepare calcium-phosphate precipitate (1ml/10cm plate)

• Transfer vector - 20µg
• Packaging plasmid - 15µg (3rd generation: pMDL g/p RRE - 10µg + pRSV-Rev - 5µg)
• Envelope plasmid - 6µg

Add water to 0.5ml, add 0.5ml 2xHBS and mix well. Add 50µl 2.5M CaCl₂ and shake briefly, keep in RT for 20-25min, add dropwise on a plate and mix gently with a medium

Change medium (6-8hrs later); remove medium with precipitate and add 6ml/plate of fresh medium

Day 4 Collection (9-10am)

• Collect medium
• Spin 3000rpm/5min/RT
• Filter through 0.45 µm

At this point virus can be used for transduction, frozen at -70°C for future use, or concentrated

Concentration

Transfer 30ml of virus to 33ml Beckman conical tubes spin at 26.000rpm/2hrs/4°C in Beckman SW28 swingle bucket rotor. After spin discard supernatant and resuspend the virus in a desired volume of serum-free medium (e.g. Cellgro or Episerf) or PBS/1% BSA, aliquot and store at -70°C. For transduction of very delicate cells the virus can be concentrated on sucrose cushion, just put 4ml of 20% sucrose on the bottom of the tube and overlay with 26ml of viral supernatant.

 

Reagents:

2 x HBS (for 500ml)
• NaCl - 8g
• KCl - 0.38g
• Na₂ HPO₄ - 0.1g
• Hepes - 5g
• Glucose - 1g Bring pH to 7.05

2.5M CaCl2

bi-distilled water