Transgenesis using retrovirus Murine Leukemia Virus (MLV)

In preparation for the first day of the retrovirus production protocol, GP2-293 cells should be thawed and passaged at least three times before the DNA transfection (Figure 1A).  Briefly, remove the cells from the liquid N2 storage tank and thaw them in a 37oC water bath making sure that the water does not touch the junction of the tube and the cap.  Once thawed pour the cells into a 15 ml tube with 4 ml of pre-warmed complete DMEM medium (DMEM with 10 % FBS and 1% of Penicillin and

Streptomycin (Pen/Strep)).  Spin the cells for 5 min at room temperature (RT) at 453 Xg (1500 rpm in the Eppendorf 5810R) and remove the supernatant by suction using a sterile Pasteur pipette.  Resuspend the cell pellet in 5 ml of complete DMEM medium.  Place 5 ml of cell suspension in a T25 tissue culture flask.  When the flask is 90-95% confluent, usually the following day, split the culture into one T75 flask (Sarstedt cell+treated flasks cat # 83.1813.302). These cells are not very adherent and the special flasks can be advantageous for this task.  Repeat this passage one more time into another single T75 flask.  After this passage expand the culture into 5 T75 flasks.  The cells from these 5 flasks will be used to seed the 60-mm plates.

Remove the medium from the T25 flask where the cells were brought up.

  1. Gently wash the cells with 5ml of 1X PBS, to remove the residual medium.  Care must be taken because the cells will lift from the flask easily. Remove the 1X PBS by suction.
  2. Add 1 ml of 0.25% Trypsin (Invitrogen cat #25200).  Let the trypsin sit on the cells for 1 min at room temperature or at 37ºC. 
  3. Add 4 ml complete DMEM medium.  Mix well with a serological pipette washing the surface of the flask thoroughly and disrupting cell clumps by pressing the tip of the serological pipette against the wall of the flask.
  4. Take 2.5 ml of cell suspension and seed into a T75 flask. 
  5. Add 12.5 ml of complete DMEM medium and incubate at 37oC, 5% CO2.
  6. Grow the cells until they are 90-95% confluent.
  7. Remove the medium from the T75 flask.
  8. Gently wash the cells with 10ml of 1X PBS to remove the residual medium.  Care must be taken because the cells will lift from the flask easily. Remove the PBS by suction.
  9. Add 3 ml 0.25% Trypsin (Invitrogen cat #25200).  Let the trypsin sit on the cells for 1 min at room temperature or at 37ºC.  This step is important not so much to get the cells off the flask as to separate them from one another. 
  10. Add 7 ml complete DMEM medium.  Mix well with a serological pipette washing the surface of the flask thoroughly and disrupting cell clumps by pressing the tip of the serological pipette against the wall of the flask.
  11. Put 2 ml into a fresh sterile T75 yellow capped flask.
  12. When cells are  90-95 % confluence, harvest the cells as above except expand into 5 flasks
  13. Distribute the 10 ml over 5 sterile T75 flasks transferring 2 ml of cell suspension to each flask.
  14. Add 13 ml complete DMEM medium to each T75 flask containing the cell suspension and incubate at 37oC with 5% CO2
  15. Grow the cells until they are 90-95% confluent then harvest for plating on 60 mm plates

Day 1: Seed the cells

  1. Remove the medium.
  2. Gently wash the cells with 10 ml of 1X PBS to remove the residual medium.  Care must be taken because the cells will lift from the flask easily. Remove the PBS by suction.
  3. Add 3 ml 0.25% Trypsin.  Let the trypsin sit on the cells for 1 min.
  4. Add 7 ml complete DMEM medium.  Mix well with a serological pipette washing the surface of the flask thoroughly disrupting cell clumps by pressing the tip of the serological pipette against the wall of the flask.
  5. Transfer the cells to a 50 ml tube and mix well.  Take a small sample, e.g. 20 µl, then mix with an equal volume of 0.4% Trypan Blue Stain and count the viable cells.
  6. Plate GP2-293 cells at a density of 2-3 X 106 cells in 60-mm plates in 5 ml of medium with serum (DMEM with 10% FBS) and 1% antibiotics Pen/Strep. Ensure that cells cover the entire surface of the plate, rocking the plate back and forth avoiding accumulation of cells at the center.  Cells should be 90-95% confluent at the time of transfection (Figure 2A).  Generally, (9) 60-mm plates are used per batch of virus, to yield 40-45 ml of non-concentrated virus.
  7. Incubate the cells overnight at 37°C, 5% CO2.

Day 2: Lipofectamine 2000 or polyethylenimine (PEI) DNA transfection.

  1. For each 60-mm plate dilute 8 µg DNA (4 µg pLNHX retroviral construct + 4 µg VSVG plasmid) in 500 µl OptiMEM (Invitrogen cat # 31985) medium (Do not add serum or antibiotics) in a sterile polystyrene tube. Mix gently. Multiply for the total number of plates.
  2. In a separate sterile polystyrene tube dilute 20 µl of Lipofectamine 2000 (Invitrogen) (1µg/µl) or PEI (1µg/µl, see below) in 500 µl of OptiMEM (no serum, no antibiotics) for each plate.  Mix gently and incubate for 5 min at RT.

Note: PEI (1 µg/ µl) – PEI is polyethylenimine 25kDa linear (Polysciences, cat no. 23966-2). To prepare stock solution:

  • Dissolve PEI in endotoxin-free dH2O that has been heated to ~80°C.
  • Let cool to room temperature.

Neutralize to pH 7.0, filter sterilize (0.22 µm), aliquot and store at -20°C; a working stock can be stored at 4°C.

  1. After the 5 minute incubation at room temperature (RT), combine the diluted DNA with the diluted Lipofectamine 2000 or PEI and mix gently.
  2. Incubate for 20 min at RT to allow the DNA-Lipofectamine 2000 or DNA-PEI complexes to form.  The solution may appear cloudy; this will not affect the transfection.
  3. While the DNA-lipid complexes are forming, remove the complete DMEM medium from the cells and replace with 2 ml OptiMEM supplemented with 10% FBS (no antibiotics).
  4. After the 20 minutes RT incubation, add 1 ml of DNA-lipid mixture drop-wise to each plate of GP2-293 cells.  Mix by gently rocking the plate back and forth. (Gentle rocking is very critical in this step. The cells need to be handled gently, especially during pipetting . If not, the cells will peel off from the plate).
  5. Incubate the cells overnight at 37oC in the 5% CO2 incubator.

Note: no differences were found between Lipofectamine 2000- and PEI-DNA transfection based on the MLV particles copy number by qPCR (Table 2  and below).

Day 3: Replace cell medium

  1. Remove the DNA/lipofectamine mixture from the GP2-293 cells and replace with 5 ml complete DMEM medium with 10% serum + 1% Pen/Strep.  Incubate at 37oC overnight. (Follow the precautions against cell damage described in Step 29) Figures 2B-D shows representative pictures of cells exposed to different DNA:PEI ratios; 1:1 (B), 1:2 (C) and 1:3 (D),  ~18 hours after transfection. The cell toxicity of PEI is evident as the higher the relative amount of PEI employed in the transfection, the higher the cell mortality observed (Figure 2B-D, yellow arrows).   

Day 4: Harvest the viral particles

  1. Harvest viral supernatant 48 to 72 hours after transfection into a 50 ml conical tube (polypropylene is fine).  Remove cell debris by low speed centrifugation (453 X g, 1500 rpm in the 5810R Eppendorf centrifuge) for 5 min at 4°C. Transfer the supernatant to a new 50 ml conical tube. Try to avoid debris which will clog the membrane in the next step. Figure 3 depicts cells that have been transfected with either lipofectamine or PEI immediately before harvesting the viral particles.
  2. Filter the supernatant through a 0.45 µm pore size PVDF membrane filter (Millipore steriflip cat # SE1M003M00)

Incubate viral supernatants with 2 Units/ml of DNase I (New England Biolabs cat #M0303L) and add CaCl2 to a final concentration of 0.5 mM.

  1. Mix gently and incubate for 3 hours at 37oC.
  2. Concentrate the virus at 50,000 X g for 90 min at 4oC (Sorvall SS-34 rotor).  Remove the supernatant. The pellet can be seen after the spin, but after you remove the supernatant it is difficult to see, so before removing the supernatant draw a circle around the pellet on the outside of the tube with a Sharpie or a Sarstedt marker.
  3. Resuspend the virus in 0.5-1% of the original volume in TNE (50 mM Tris, 130 mM NaCl, 1 mM EDTA, pH 7.8) or schistosomule medium (Suttiprapa et al. 2012b) at 4oC overnight (tilt the tubes so the liquid covers the pellet).
  4. Aliquot and use fresh virus (recommended) or store at -80oC (snap freeze).

Note: From Day 3 onwards, materials that have come in contact with the samples must be treated with 20% bleach (Clorox) or similar disinfectant to inactivate viruses/virions associated with the procedure. Similarly disinfect supernatants to be discarded, disposable plasticware, serological pipettes, etc.  The culture cabinet and other equipment that has been in contact with the virus must be cleaned and exposed to UV for 30 min.