University of South Alabama

  1. Production of retrovirus-containing supernatants
  2. Production of lentivirus-containing supernatants
  3. Titration of retro- and lentiviral vectors
  4. Infecting target cells with retro- and lentiviruses
  5. Concentration of lentiviral vectors

Preparing Retrovirus using Phoenix Lines (adapted from Gary Nolan Lab protocol)

Phoenix cell lines are available from ATCC Safe Deposit. The Phoenix cells are not listed in ATCC's regular catalogue. To order these cells, please send your request by email to: sales@atcc.org. An ATCC Safe Deposit representative will contact you back with ordering instructions. 

Gene Delivery Core can not supply packaging cell lines due to restrictions imposed by MTA.

IMPORTANT NOTE

The viral supernatants produced by these methods might,depending upon your retroviral insert, contain potentially hazardous recombinant virus. The user of these systems must exercise due caution in the production, use and storage of recombinant retroviral virions, especially those with amphotropic and polytropic host ranges. This consideration should be applied to all genes expressed as amphotropic and polytropic pseudotyped retroviral vectors. Appropriate NIH and other regional guidelines should be followed in the use of these recombinant retrovirus production systems. The user is strongly advised NOT to create retroviruses capable of expressing known oncogenes in amphotropic or polytropic host range viruses.

 

Phoenix Retroviral Producer Line Protocol

Day 0: Preparation of Phoenix Retrovirus Producer cells for Transfection.

  • 18-24 hours prior to transfection, plate Phoenix cells at 1.5-2 million cells per 6 cm plate in producer cell growth media.
  • After adding cells, set plates in incubator and gently shake forward and backward, then side to side, 3-4 times. This distributes cells evenly about the plate. Do not disturb cells for several hours while they attach to plate.
  • Cells are allowed to attach for 10 hours to 24 hours on 10 or 15 cm plates.
  • At 2/3 confluence, a 10 cm plate should provide 5 million cells, a 15 cm plate will contain about 7.5-15 million cells. It is at this subconfluent stage that cells are most transfectable and will survive the rigours of transfection best, giving the highest titer virus possible.
  • Media: DMEM, 10% FCS, 1% Penicillin-Streptomycin, 1% Glutamine.
  • Passaging Phoenix cells:
    • Never let cells reach confluence. This will reduce transfection efficiency in the short term. For maximally healthy cells a split of 1:4 or 1:5, of a 70-80% confluent 10 cm plate, into a new plate every 2-3 days should provide optimal cell conditions. Local media and serum conditions will vary.
    • Passage of PhoenixTM cells every few months in Hygromycin (at 300 ug/ml) and Diphtheria fToxin (1 ug/ml) for one week is recommended. Cells can be analyzed and sorted by FACS for expression of CD8 (a proxy measure of gag-pol in this cell line) and for surface expression of envelope protein with 83A25 antibody (see Chesboro et al (ref)).
    • Two million cells on a 60 mm plate is a good starting point for seeding Phoenix cells prior to transfection; it is important to titer slightly up and slightly down to maximize transfection efficiencies. Efficiencies of 50-60% as determined by X-gal staining of the Phoenix cells should be achieved. We have found that the highest transfection efficiencies are obtained with Phoenix cells that are 70-80% confluent at the time of transfection. Day 1: Transfection
      • Prepare the DNA in HBS for application to cells. 1. About 5 minutes prior to transfection, add chloroquine to each plate to 25uM (chloroquine stock is 50 mM; for 3 mL media + 1 mL DNA, add 2u1).
        • Chloroquine acts to inhibit lysosomal DNases by neutralizing vesicle pH.
        • DNA delivered by Ca2PO4 transfection is thought to transit through lysosomes. 
      • To a 15 mL tube, add (per 6 cm plate, with all reagents at room temperature):
        • 5-10 ug DNA (DNA is added in a drop to side of tube).
        • 438 u1 dd H2O (wash the DNA to bottom of tube with water).
        • 61 ul 2M CaC12 (from Mallinkrodt) -mix thoroughly with finger tapping.
        • 500 ul total volume.
        • Scale volume and DNA/reagent amounts if necessary.  
      • Add 0.5mL 2xHBS quickly then bubble vigourously with automatic pipettor (keep eject button depressed) for 3 - 15 sec (actual length of bubbling time depends on each batch of 2xHBS).
      • Add HBS/DNA solution dropwise onto media (gently and quickly) by spreading across cells in media.
      • Observe the cells under a microscope; you should observe evenly distributed VERY small black particles.
      • Put plate(s) in 37 C incubator; rock plates forward and backward/back and forth a few times to evenly distribute DNA/CaPO4 particles.
      • HEPES for Calcium Phospahte Coprecipitation Transfection
        • 1. Make a stock solution of Na2HPO4 dibasic (5.25 g in 500 ml of water)
        • 2. Make 2 x HBS: 8.0 g NaCl 6.5 g HEPES (sodium salt) 10 ml Na2HPO4 stock solution
        • 3. pH to 7.0 using NaOH or HCl. Bring volume up to 500 mls. Check pH again. The pH is very important, it must be exactly 7.0  
      • Additional Notes:
        • 1. HEPES is from Sigma (catalog # H-7006)
        • 2. CaCl2 is from Mallinkrodt (catalog # 4160)
        • 3. Because pH is so important make 3 batches pH 6.95, pH 7.00, pH 7.05. Test each solution and use the one that yields the best precipitate.
        • 4. All reagents should be at room temperature prior to use

Day 1: Transfection

  • Prepare the DNA in HBS for application to cells. 1. About 5 minutes prior to transfection, add chloroquine to each plate to 25uM (chloroquine stock is 50 mM; for 3 mL media + 1 mL DNA, add 2u1).
    • Chloroquine acts to inhibit lysosomal DNases by neutralizing vesicle pH.
    • DNA delivered by Ca2PO4 transfection is thought to transit through lysosomes. 
  • To a 15 mL tube, add (per 6 cm plate, with all reagents at room temperature):
    • 5-10 ug DNA (DNA is added in a drop to side of tube).
    • 438 u1 dd H2O (wash the DNA to bottom of tube with water).
    • 61 ul 2M CaC12 (from Mallinkrodt) -mix thoroughly with finger tapping.
    • 500 ul total volume.
    • Scale volume and DNA/reagent amounts if necessary.  
  • Add 0.5mL 2xHBS quickly then bubble vigourously with automatic pipettor (keep eject button depressed) for 3 - 15 sec (actual length of bubbling time depends on each batch of 2xHBS).
  • Add HBS/DNA solution dropwise onto media (gently and quickly) by spreading across cells in media.
  • Observe the cells under a microscope; you should observe evenly distributed VERY small black particles.
  • Put plate(s) in 37 C incubator; rock plates forward and backward/back and forth a few times to evenly distribute DNA/CaPO4 particles.
  • HEPES for Calcium Phospahte Coprecipitation Transfection
    • 1. Make a stock solution of Na2HPO4 dibasic (5.25 g in 500 ml of water)
    • 2. Make 2 x HBS: 8.0 g NaCl 6.5 g HEPES (sodium salt) 10 ml Na2HPO4 stock solution
    • 3. pH to 7.0 using NaOH or HCl. Bring volume up to 500 mls. Check pH again. The pH is very important, it must be exactly 7.0  
  • Additional Notes:
    • 1. HEPES is from Sigma (catalog # H-7006)
    • 2. CaCl2 is from Mallinkrodt (catalog # 4160)
    • 3. Because pH is so important make 3 batches pH 6.95, pH 7.00, pH 7.05. Test each solution and use the one that yields the best precipitate.
    • 4. All reagents should be at room temperature prior to use

Days 2-3: 24-48 hours post-transfection

  • Collect media and filter through 0.45 um cellulose acetate filter. Store frozen.
  • Overlay cells with 5 ml of fresh DMEM.
  • Repeat at 36 and 48h after transfection

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Protocol for Production of Lentiviral Vectors in 293T cells (pdf)

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 - µg)
  • 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
  • Na2HPO4 - 0.1g
  • Hepes - 5g
  • Glucose - 1g Bring pH to 7.05

2.5M CaCl2
bi-distilled water

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Titration of Retro- and Lentiviral Vectors Encoding Fluorescent Protein(s)

Day 1 (9-12am)

  • plate 30.000 293t per well in 24 well plate in 1 ml medium/well

Day 2 (4-6pm)

  • count cells in 1 well (should have 60-80.000)
  • transduce the cells with 6, 4-fold serial dilutions in 250µl total vol; take 50µl for non-conc. or 1µl for conc. vector stock for as a first dilution

Day 3 (9-12am)

  • add 1ml of medium

Day 5

  • split cells and analyze fluorescence by FACS and read percentage from linear values (usually 5-10%) Titer is a number (percentage) of cells transduced by a given vol and counted on D2 e.g. 1µl gives you 10% of positive cells and you had 50.000 cells on D2, so you have 5.000TU/µl --> 5x106 TU/ml

Day 7-8

  • isolate DNA for QPCR

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Infecting Target Cells

  1. Plate the target cells 12-18 hr before infection, at a cell density of 1-2 x 105 per 60-mm plate.
    If you will be using infected cells for a biological assay, ensure that the control cells are treated with an insert-free virus under identical conditions.
    Note: The viral pre-integration complex enters the nuclei of actively dividing cells only.

  2. For infection, collect medium from packaging cells and filter medium through a 0.45-µm cellulose acetate or polysulfonic (low protein binding) filter. Do not use a nitrocellulose filter because it binds proteins in the retroviral membrane and destroys the virus.

  3. Add virus to target cells. Until you have determined the viral titer, use as much virus-containing medium as possible for the infection. Store remaining viral supernatant at -80°C.
    Notes:
    • Titer will decrease ~2-4-fold per freeze-thaw cycle.

      The optimal final concentration of polybrene may need to empirically determined but generally falls within a range of 2-12 µg/ml.

      Excessive exposure to polybrene (>24 hr) can be toxic to cells. Alternatively, perform infections sequentially, ~12 hr apart. Doing so increases the efficiency of infection, but also increases copy number. Cellular receptors can be occupied by soluble envelope and/or nonfunctional virions. Therefore, to ensure that cellular receptors will be unoccupied by viral envelope, allow cells to rest for a minimum of 12 hr between each infection.

  4. Add polybrene to a final concentration of 4-8 µg/ml.

  5. Replace medium with fresh medium after 24 hr of incubation.

  6. To determine the efficiency of infection, subject a small subpopulation of cells to antibiotic treatment. The infected cells should be used for experiments or for selection as soon as possible, but not earlier than 24 hr after the last infection.
    The growth of some target cells is strongly affected by media conditioned by the packaging cells. You can take certain precautions to avoid an adverse effect induced by the packaging cell-derived supernatants:
    • Dilute virus-containing media at least 2-fold with fresh medium.
    • Expose target cells to the virus for 4-6 hr and then replace with fresh medium.

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