Phoenix - Helper dependent protocol

Preparing Retrovirus using Phoenix Lines. This is one of two transfection protocols you can find in this web page set.

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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.

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Phoenix Retroviral Producer Line Protocol

{Day 0 | Day 1 | Day 2 | Day 3 | Day 4 | Day 5 | X-Gal Staining}

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 Phoenix™ cells every few months in Hygromycin (at 300 ug/ml) and Diptheria Toxin (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.

   

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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 foreward 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.

   

• Please e-mail any questions to Angelica Trejo: angtrejo@stanford.edu

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Day 2: 24 hours post-transfection

• Detoxify media and prepare target cells
  • Change media to 3 mL fresh DMEM, 10% FCS
  • Virus is more stable if incubation is carried out at 32 Celsius, although 37 is okay.
  • Do not leave chloroquine on cells more than 24 hours. It is toxic.
  • Prepare for Titering if necessary
• Split NIH 3T3 cells at 200,000 per 6 cm plate in DMEM, 10% CS (1% PentStrep, 1 % Glut).
• If using suspension cells, they should be growing in log phase at time of infection (see below--for Jurkats, ideal density at time of infection is 5E+5 /mL).

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Day 3: 48 hours post-transfection - infecting cells with retroviral supernatent

• Pipette supernatant from transfected Phoenix cells into 15 mL tubes and centrifuge at 1500 rpm for 5 minutes to pellet cell debris.
  • Filtering through 0.45 um filter removes cells as well.
  • Supernatant can be frozen at -80 C for later infection, although titer drops by one-half for each freeze-thaw cycle.
• Stain Phoenix cells with X-gal to gauge transfection efficiency
  • This only works if you used a lacZ or other stainable reporter virus).
  • If your virus does not contain such a marker, you can dope the transfection with about 1/10 molar plasmid of a marker that will not interfere with your experiment).
• Remove 1 mL media from each 3T3 plate.
• Add 3 uL polybrene (polybrene is lOOOx at 5 mg/ml) to each 3T3 plate; with gentle and thorough shaking.
• Adherent Cells:
  • Add 1 mL viral supernatant to each 3T3 plate and place at 32/37 Celsius with gentle shaking.
  • Incubate for 8-24 hours, spin cells, and wash away virus supernatent.
• Suspension cells:
  • Pellet 5x10^5 suspension cells, resuspend cell pellet in 1 mL virus + 1 uL lOOOx polybrene.
  • Suspension cells, especially some B cells and T cells are sensitive to polybrene
  • It may be necessary to titrate polybrene to lower levels.

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Day 4: Remove Virus Supernatent.

• 24 hours post-infection: change media on 3T3s to fresh DMEM, 10% CS and place at 37 Celsius.
  • For suspension cells, spin out of media and resuspend in 2 mL fresh media.
• Place at 37 Celsius.

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Day 5: 24- 48 hours post-infection

• Cells are now ready to assay for biochemical event of interest.
  • The actual reverse transcription and integration take place within 24-36 hours, depending on cell growth kinetics.
  • Expression can start to be observed at 24 hours, usually maxing out at 48 hours.
  • From there on in continued retroviral expression might drop over a period of weeks to months, depending on cell line, site of integration, relative toxicity of insert and a whole bunchof things nobody really understands.
• Your on your own from here. However, if you did add a reporter enzyme, such as lacZ, you can stain 3T3s or supsension cells with X-gal or prepare them for FACS-Gal.

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X-gal Staining

• Prepare Solutions:
  • Fixative
    • PBS/0.05%
    • Glutaraldehyde (Glutaraldehyde is 25% stock/500x, from Sigma)
  • Staining solution
    • per 3 mL ferri/ferrocyanide solution
      • 40-50 uL X-Gal (40mg/mL in DMSO--store at -20oC in the dark).
    • 25X ferricyanide solution:
      • 300mM [K3Fe(CN)6]
      • 130mM MgCl2 in ddH2O
    • 25X ferrocyanide solution:
      • 300mM [K4Fe(CN)6]
      • in ddH2O.
    • Protect the solutions from exposure to light and store at 4oC.
  • Washing solution
    • PBS
    • For nonadherent cells add FCS to 1-5%
• Remove media from adherent cells or spin down nonadherent cells in 15mL conical tube.
• Add 2 mL fixative to 6cm plate of adherent cells or resupsend nonadherent cells in 1 mL fixative. Leave for 1 min.
  • For adherent cells, remove and wash 3xPBS (first two washes are quick, third is for 3 min).
  • For nonadherent cells, quench fixative by adding 5-lOmL PBS/1-5% FCS to conical and spin down again.
• Layer 3mL staining solution onto adherent cells or resuspend nonadherent cells in 1 mL staining solution and place in well of 24well plate.
• Optimal staining will occur 24 hours later. If longer, remove staining solution from cells at 24 hours and re-layer/resupsuspend in ferri/ferrocyanide solution without X-Gal.
• Milder Fixative:
  • 2% Paraformaldehyde/0.2% Glutaraldehyde
  • Should be left on cells for 2 min and washed 3xfast with PBS.
  • It is much more difficult to prepare:
    • 1. 4% paraformaldehyde stock: in fume hood, dissolve 8g powder in 150mL of 0.1M sodium phosphate pH 7.3 (66mM NA2HPO4 or 33mM NAH2PO4), stirring and heating at 60oC
    • Add 10N NaOH at rate of 1 drop/min until solution clears. Bring up volume to 200mL with 0.1 M sodium phosphate pH 7.3. Store at 4 C for up to 1 month.
    • Combine 50mL 4% paraformaldehyde solution with 49.2 mL 0.1M sodium phosphate pH 7.3 and 0.8mL 25m% glutaraldebyde. Store at 4 for up to 1 week.