An Efficient Method for Adenovirus Production

Safety Note: In general, adenoviruses are classified as biosafety level 2 organisms and thus, all manipulations must be done in a Class II biosafety cabinet by a trained person, wearing biohazard protective equipment (including gloves, face-mask for biological aerosols, lab coat, etc.). All solid materials contaminated with the adenovirus must be disinfected with a 10% bleach solution for 30 min and autoclaved for 30 min at 121 °C and 1 bar. Depending on the gene inserted, the adenovirus created may have dangerous potential and may be classified in other biosafety levels.

1. Experimental preparation

1. Use a separate cell culture hood for adenoviral manipulations, and a separate incubator for each adenovirus type. Use T flasks with filter caps for viral packaging and amplification; avoid as much as possible transduction experiments in vented Petri plates.
2. Empty the cell culture hood after each use and expose it to UV for 15 min.
3. Autoclave periodically the pipette aid, pipettes, and other utensils. If possible, culture in a separate cell culture laboratory/hood the cells for adenoviral packaging (AD293 cells) and the cells to be used in transduction experiments. Batches of different adenoviruses amplified in the same period should be checked for cross-contamination by PCR.
4. Prepare the following solutions.
   1. Prepare SOB (Super Optimal Broth) medium: 20 g of tryptone, 5 g of yeast extract, 0.5 g of NaCl (10 mM final concentration), 2.5 mL of 1 M KCl (2.5 mM final concentration), amd H₂O to 1 L. After autoclaving at 121 °C, add the following sterile solutions: 5 mL of 1 M MgCl₂ and 5 mL of 1 M MgSO₄.
   2. Prepare SOC (Super Optimal broth with Catabolite repression) medium: in 1 L sterile SOB add the following sterile solutions: 20 mL of 1 M glucose, 5 mL of 1 M MgCl_2, and 5 mL of 1 M MgSO₄.
   3. Prepare precipitation solution: dissolve 29.5 g of potassium acetate in 60 mL of H₂O, add 11.5 mL of acetic acid, and H₂O to 100 mL.
   4. Prepare resuspension buffer: 95 mL of 20% glucose, 5 mL of 1 M Tris-Cl pH 8, 4 mL of 0.5 M EDTA pH 8, and add H₂O to 200 mL.
   5. Prepare lysis solution: 4.8 mL of 8.3 M NaOH, 10 mL of 20%SDS, and H₂O to 200 mL.

2. Recombination of pAdTrack-GFP viral vector with pAdEasy-1 plasmid in BJ5183 bacteria

1. Linearization of pAdTrack-GFP and purification of the linearized plasmid.
   1. Prepare the following digestion mix on ice:
      10 µg of pAdTrack-GFP
      5 µL of 10x colorless buffer
      2 µL of Pme I
      H₂O of to a final volume of 50 µL.
   2. Incubate at 37 °C for 3 hours in a water bath.
   3. Inactivate at 65 °C for 20 min.
   4. Check the efficiency of the digestion of pAdTrack-GFP with Pme I: run 1 µg of the digested plasmid in parallel with 1 µg of undigested plasmid on a 0.8% agarose gel.
2. Isolation and purification of DNA
   NOTE: Steps 1-6 need to be performed in a fume hood.
   1. Add an equal volume of phenol/chloroform/isoamyl alcohol (25:24:1) over the digestion mixture and invert the tube until the mixture is homogeneous.
   2. Centrifuge for 3 min at 16,200 x g, and then transfer the upper aqueous phase to a collection tube.
   3. Add an equal volume of phenol/chloroform/isoamyl alcohol (25:24:1) over the lower organic phase and vortex.
   4. Centrifuge for 3 min at 16,200 x g, and then transfer the upper phase to the same collection tube.
   5. Add an equal volume of chloroform over the aqueous phase harvested in the collection tube and vortex.
   6. Centrifuge for 3 min at 16,200 x g, and then transfer the upper aqueous phase to a new collection tube.
   7. Add a 1/10 volume of 3 M sodium acetate, and 2 volumes of cold 100% ethanol and vortex.
   8. Incubate for 1 hour at -70 °C or overnight at -20 °C.
   9. Thaw the sample on ice and centrifuge it for 10 min at 16,200 x g and 4 °C.
   10. Remove the supernatant and add 750 µL of 75% ethanol.
   11. Centrifuge for 3 min at 16,200 x g and 4 °C and remove the supernatant.
   12. Briefly spin the tube to remove all the supernatant and dry the pellet in the hood. Do not dry the DNA pellet for a long time because it is tricky to dissolve.
   13. Dissolve the pellet in 15 µL of H₂O.
   14. Measure the DNA concentration using a spectrophotometer (e.g., Nanodrop).
3. Transformation of AdEasier-1 bacteria with pAdTrack-GFP
   NOTE: In this step, the recombination of pAdTrack-GFP with pAdEasy-1 plasmid takes place.
   1. Prepare AdEasier-1 (BJ5183-containing pAdEasy-1, Addgene #16399) chemical competent bacteria, using a commercial transformation kit, following the manufacturers’ instructions. Keep aliquots of 100 µL competent bacteria at -80 °C.
   2. Thaw an aliquot of competent AdEasier-1 bacteria on ice and add 1 µg of purified Pme I -digested pAdTrack-GFP. Mix gently by flicking the tube (do not pipette the mixture). Incubate for 10 min on ice.
   3. Add 900 µL of SOC medium and incubate for 1 hour at 37 °C with shaking.
   4. Microfuge for 5 min at 600 x g.
   5. Remove 900 µL of the supernatant, mix the pellet and the supernatant, and seed the transformed bacteria on LB-agar plates with kanamycin.
   6. Incubate for ~16 hours at 37 °C (do not exceed 18 hours).
4. Selection of the possible positive clones by PCR
   1. Divide toothpicks into two halves and sterilize the half-toothpicks by autoclaving.
   2. Pick up small and translucent colonies using sterile half-toothpicks.
   3. Briefly, rotate the half-toothpick with bacteria in 10 µL water (in a PCR tube) and then put the half-toothpick in a 1.5 mL Eppendorf tube containing 100 µL SOC medium with kanamycin. Incubate for 4-6 hours at 37 °C, while you test the clones by “negative” and “positive” PCR.
   4. Incubate the PCR tubes containing 10 µL water with bacteria for 5 min at 95 °C to obtain the bacterial sample and run in parallel the “negative” and “positive” PCR.
   5. “Negative” PCR – to test the pAdTrack-GFP integrity: Prepare the following PCR mix for the negative PCR on ice.
      5 µL of the bacterial sample
      0.1 µL of primer Forward (4631 F: 5’-CAGTAGTCGGTGCTCGTCCAG)
      0.1 µL of primer Reverse (5616 R: 5’-TATGGGGGCTGTAATGTTGTCTC)
      0.1 µL of dNTP 10 mM
      3 µL of 5x Buffer
      1.5 µL of MgCl₂ 25mM
      0.1 µL of GoTaq Polymerase
      H₂O to a final volume of 15 µL
      NOTE: The positive control in which the DNA template is the pAdTrack-GFP vector must be included.
   6. “Positive” PCR – to test the presence of the gene of interest. Use specific primers for the inserted gene and prepare the mix as in the previous step. The primers used for GFP were the following:
      F: 5’-CAAGGACGACGGCAACTACA
      R: 5’-ATGGGGGTGTTCTGCTGGTA
   7. Run in parallel the “negative” and the “positive” PCR. The PCR Program is: 5 min, 95 °C; 40 cycles of the following steps: 30 sec, 95 °C; 30 sec, 68 °C; 1 min, 72 °C; final elongation: 10 min, 72 °C.
      NOTE: Adapt the annealing temperature for amplification the gene of interest.
   8. Evaluate the PCR products on a 1% agarose gel and make the selection of the clones.
   9. Consider for further processing the clones that give no PCR products for the “negative PCR” and the specific PCR product after the “positive PCR”.
5. Grow the bacterial cultures of selected recombinant clones
   1. Dilute the cultures of the presumed positive clones (resulted in the step 2.4.3.) in 4 mL of SOC medium with kanamycin, and incubate them overnight at 37°C with shaking.
6. Isolation of plasmid DNA from AdEasier-1 bacteria (Miniprep using alkaline lysis)
   1. Transfer 1.5 mL of bacterial culture in microcentrifuge tubes, centrifuge for 1 min at 16,200 x g, and remove the supernatant.
   2. Transfer another 1.5 mL bacterial culture in the same tube, repeat centrifugation, and remove the supernatant.
   3. Add 200 µL of resuspension buffer (50 mM glucose, 10 mM EDTA, 25 mM Tris-HCl pH 8).
   4. Add 200 µL of lysis solution (0.2 N NaOH, 1% SDS), mix gently by inverting the tube.
   5. Add 200 µL of precipitation solution (60 mL of 5 M potassium acetate, 11.5 mL of glacial acetic acid, add H₂O until 100 mL), and mix gently by inverting the tube.
   6. Centrifuge for 3 min at 16,200 x g.
   7. Transfer the supernatant in a new microcentrifuge tube, add 500 µL isopropanol, mix and incubate for 20 min on ice.
   8. Centrifuge for 15 min at 16,200 x g and add 500 µL of 75% ethanol.
   9. Centrifuge for 10 min at 16,200 x g and remove the supernatant.
   10. Centrifuge for 3 min at 16,200 x g, remove the supernatant and add 15 µL of H₂O.
7. Amplification, isolation, and re-testing of the recombined plasmids
   1. Transformation of DH5α bacteria with the DNA isolated from AdEasier-1 cells.
      1. Prepare DH5α competent bacteria using the commercial transformation kit, following the manufacturers’ instructions.
      2. Thaw 100 µL of DH5α competent bacteria on ice, add the recombinant DNA, and incubate 10 min on ice. Then seed the bacteria onto LB-agar plates with kanamycin.
      3. Incubate at 37 °C overnight.
      4. Pick-up several colonies and grow each in 2 mL of LB medium with kanamycin, at 37 °C, overnight, with shaking.
   2. Isolate the DNA (Miniprep using alkaline lysis) and resuspend the obtained DNA in 25 µL H₂O.
   3. Confirm the positive clones by enzymatic digestion.
      1. Prepare the following mix on ice:
         5 µL of recombinant DNA
         1.5 µL of 10x colorless Buffer
         0.5 µL of Hind III or Pst I
         H₂O to a final volume of 15 µL
      2. Incubate at 37 °C for 30 min.
         NOTE: As a control, digest also the pAdTrack-GFP and pAdEasy-1 plasmids.
      3. In each sample add 3 µL of Sx6 loading buffer with RNase A (if RNase A is not present in the miniprep buffers).
      4. Run the digested DNA fragments on 1% agarose gel electrophoresis.
         NOTE: The digestion pattern of a positive clone includes the majority of fragments of the digested pAdEasy plasmid, revealing pAdEasy recombination with the pAdTrack vector. The gene of interest should be evidenced by digestion with the restriction enzymes used for cloning.
   4. Preparation of plasmid DNA (transfection-grade) for adenovirus packaging.
      1. Grow a 200 mL culture of bacteria from a positive clone to isolate the plasmid DNA.
      2. Isolate the plasmid DNA using a commercial kit for plasmid DNA Midiprep (e.g., Qiagen Plasmid Midi Kit) following the manufacturer’s instructions.

3. Packaging the adenoviral particles

1. Day 1. Seed the AD293 cells
   1. Wash the AD293 cells with PBS and incubate them with 0.125% Trypsin for 2-5 min at 37 °C.
   2. Collect the cells in cold medium with serum.
   3. Centrifuge for 5 min at 400 x g at 4 °C.
   4. Resuspend the cells in medium with serum and seed the cells at a density of ~2 x 10⁶/T25 flask. Preferably, use a flask with a filter.
2. Day 1. Digest the recombinant DNA with Pac I
   1. Prepare the following mix:
      6 µL of recombinant DNA (1 µg/µL)
      2 µL of Pac I
      2.5 µL of 10x colorless Buffer
      H₂O to a final volume of 25 µL
   2. Incubate for 3 h (or overnight) at 37 °C, and then inactivate the enzyme at 65 °C for 20 min.
   3. DNA precipitation with ethanol: add 2.5 µL of (1/10 v/v) 3 M sodium acetate and 2-3 volumes of 100% ethanol. Incubation for 30 min at -70 °C or overnight at -20 °C.
   4. Centrifuge at 16,200 x g for 30 min at 4 °C and resuspend the pellet in sterile water.
3. Day 2: Transfection of AD293 cells using K2 reagent
   1. Add 40 µL of K2 Multiplier over the cells, two hours before transfection.
   2. Prepare A and B solutions:
      Solution A: Add 6 µg of Pac l-linearized DNA in 260 µL of Opti-MEM.
      Solution B: Add 21.6 µL of K2 Reagent in 248.4 µL of Opti-MEM.
   3. Add solution A over solution B and mix gently by pipetting.
   4. Incubate the mixture for 20 min at room temperature. Add dropwise A and B mix to the cells.
4. Day 3-11: Monitor the GFP expression by fluorescence microscopy
   NOTE: Cells should appear green in fluorescence microscopy and should gradually detach.
5. Day 11: Harvest the F1 adenoviral particles
   1. Collect the detached cells and the medium in a 50 mL tube, scrape the adherent cells, and add them in the same tube.
   2. Centrifuge for 5 min at 400 x g, collect the supernatant in a new tube and resuspend the cell pellet in 0.5 mL of PBS.
   3. Cell disruption
      1. Transfer the cell suspension in a microcentrifuge tube.
      2. Perform three freeze/thaw cycles (freeze in liquid nitrogen or at -80 °C /thaw at 37 °C for maximum 7 min).
      3. Pass the broken cells through a 23 G syringe needle three times.
   4. Remove the cell debris by centrifugation at 9,600 x g for 12 min.
   5. Transfer the supernatant to the 50 mL tube with the collected medium.

4. Amplification of the adenovirus

NOTE: If the AD293 cells did not reach the necessary confluence, the aliquots of the adenoviral stocks (lysate obtained from the virus-producing cells) to be used for infection may be stored at -80 °C.

1. Prepare the F2 adenoviral particles.
   1. Seed the AD293 cells in a T75 flask (5 x 10⁶ cells/flask).
   2. Infect ~90% confluent AD293 cells using the F1 adenoviral particles: add the cell homogenate and the medium from the T25 flask over the cells grown in the T75 flask.
   3. Monitor the GFP expression by fluorescence microscopy.
   4. Harvest the virus-producing cells when ~90% of the transduced AD293 are detached (~ the 5^th day after transduction). Keep the cell culture medium at 4 °C.
   5. Disrupt the cells (similarly with those for F1) in 1 mL PBS.
2. Prepare the F3 adenoviral particles.
   1. Infect ~90% confluent AD293 cells seeded in T175 flask with F2 adenoviral particles and the cell culture medium from the F2 adenoviral particles.
   2. Harvest the cells (~5 days after transduction).
   3. Disrupt the cells (similarly with those for F1) in 2 mL of PBS.
3. Prepare the F4 adenoviral particles.
   1. Infect 5 T175 flasks containing ~90% confluent AD293 cells with F3 adenoviral particles and cell culture medium.
   2. Harvest the cells (~5 days after transduction).
   3. Disrupt the cells (similarly with those for F1) in 3 mL of PBS.
4. Prepare the F5 adenoviral particles.
   1. Infect 25 T175 flasks containing ~90% confluent AD293 cells with F4 adenoviral stock and cell culture medium.

5. Purification of the adenovirus from cell lysate and culture medium

1. Harvesting the virus-producing cells and the culture medium.
   1. Harvest the AD293 cells of F5 after 5 days from transduction.
   2. Save the medium in a sterile bottle for precipitation of the adenoviral particles.
      NOTE: Keep the medium in the refrigerator until purification of the adenovirus.
   3. Centrifuge the cells at 400 x g, for 5 min, at 4 ^oC.
   4. Resuspend the final pellet in 5 mL of 10 mM Tris HCl, pH 8 with 2 mM MgCl₂.
   5. Aliquot the suspension in 1.5 mL tubes.
   6. Disrupt the cells (similarly with those for F1): three cycles of freezing/thawing.
      NOTE: If the ultracentrifugation cannot be performed immediately, keep the samples at -80 °C.
   7. Pass the cell suspension through a 23G syringe needle for three times.
   8. Centrifuge the homogenate at 9 600 x g, for 12 min.
   9. Save the supernatant for adenovirus purification by CsCl gradient ultracentrifugation.
2. Precipitation of the adenovirus released in the culture medium.
   1. Bring the bottle with saved cell culture medium at room temperature.
   2. Add 121 g ammonium sulfate to every 500 mL of cell culture medium (saturation of the solution should be between 40 – 42%).
   3. Mix carefully until the ammonium sulfate is completely dissolved.
   4. Incubate for a minimum of 2.5 hours at room temperature.
   5. Centrifuge at 1600 x g, for 15 min, at 22 ^oC and save the pellet.
   6. Resuspend the pellet in 4 mL of 10mM Tris HCl pH 8 with 2mM MgCl₂; this suspension should be purified immediately by CsCl gradient ultracentrifugation.
      NOTE: If the purification step cannot be subsequently performed, dialyze overnight the resuspended pellet against 10mM Tris HCl, pH 8 with 2mM MgCl₂.
3. Adenovirus purification by ultracentrifugation.
   1. Prepare a discontinuous CsCl gradient in polypropylene tubes for SW41Ti rotor. Add 3 mL of 765 mg/mL CsCl (high density: 1.4 g/L) at the bottom of the tube. Slowly add 3 mL of 288.5 mg/mL CsCl (low density: 1.2 g/L) on top of the first CsCl layer.
   2. Gently overlay 3 – 4 mL of adenoviral particle suspension released from the cells or precipitated from the cell culture medium (as described before) on top of the gradient.
   3. Fill the tubes with mineral oil, and put the tubes in the cold SW41Ti buckets.
   4. Equilibrate the tubes. Make sure that the filled polypropylene tubes are loaded symmetrically into the rotor. Put the rotor in the ultracentrifuge.
   5. Centrifuge at 210,000 x g and 4 °C, for 18 hours, no brake.
   6. Place the ultracentrifuge tubes on a stand with a black paper behind to get the bands.
   7. Discard the clear upper phase, the cell debris, and the upper band in a waste container with the bleaching solution.
   8. Harvest the lowest band that contains the complete adenovirus (~700 µL – 1 mL) in a sterile 1.5 mL tube and keep it on ice.
   9. Pre-wet a dialysis cassette in dialysis buffer (10 mM Tris-Cl buffer pH 8, 2 mM MgCl₂).
   10. Inject the purified adenovirus into the dialysis cassette using a 2 mL syringe.
   11. Dialyze overnight against 10 mM Tris-Cl buffer pH 8, 2 mM MgCl₂ (change the dialysis buffer 3 – 4 times).
   12. Harvest the adenoviral stock from the dialysis cassette in aliquots of 10 – 100 μL.
   13. Add sucrose to 4% final concentration to viral aliquots (for cryoprotection).
   14. Store aliquots at -80 °C.

6. Adenovirus titration

1. Day 1: Plating the cells
   1. Seed the AD293 cells at a density of 2.5 × 10⁵ cells per well (in a 12-well culture plate) in 1 mL complete growth medium, as shown in Figure 2. Ensure that cells are spread evenly in each well for accurate titer determination.

Figure 2: Titration plate design. Please click here to view a larger version of this figure.

2. Day 2: Transduction of cells
   1. Detach the cells from one well with trypsin and count them. Note this number because it will be used to calculate the viral titer.
   2. Perform serial dilutions (1/10⁴; 1/10⁵; 1/10⁶; 1/10⁷) of the viral stock in 1 mL of complete growth medium as follows:
   3. 1/10³: dilution of the virus stock – add 2 μL of viral stock to 1998 μL of complete medium.
   4. 1/10⁴: Make 1:10 dilution of 1/10³ by diluting 120 μL to 1080 μL of complete medium (A).
   5. 1/10⁵: Make 1:10 dilution of B by diluting 120 μL of A to 1080 μL of complete medium (B).
   6. 1/10⁶: Make 1:10 dilution of C by diluting 120 μL of B to 1080 μL of complete medium (C).
   7. 1/10⁷: Make 1:10 dilution of D by diluting 120 μL of C to 1080 μL of complete medium (D).
      NOTE: Prepare 3 tubes of each dilution (A, B, C, D) to perform the experiment in triplicates.
   8. Remove the cell culture medium from the wells and add the prepared dilutions of the virus, as shown in Figure 2.
3. Day 3: Monitoring GFP expression
   1. Check the wells for the presence of green cells using a fluorescence microscope.
4. Day 4: Flow cytometry analysis of GFP-positive cells
   1. Prepare and label twelve 1.5 mL tubes.
   2. Collect the cell culture medium (together with the detached cells) in 1.5 mL tubes and keep them on ice.
   3. Add 200 μL of trypsin in each well.
   4. Incubate the plate for 2 – 3 min at 37 °C in the CO₂ incubator.
   5. Harvest the cells in the same Eppendorf tubes with the cell culture medium. Keep the tubes on ice.
   6. Pellet the cells at 400 x g, for 5 min, at 4 °C.
   7. Remove the supernatant; keep the tubes on ice.
   8. Resuspend the pellet in 250 μL of PBS + 2% FBS; keep the tubes on ice.
   9. Transfer the cell suspension in flow cytometry tubes or plate.
   10. Run the samples on a flow cytometer recording the fluorescence of the GFP expressing cells.
       Titer calculation: The samples with 5 – 20% GFP positive cells from the parent population should be taken into account for the calculation of viral titer using the following formula:
       Titer (TU/mL) = D x F/100 x C/V
       D = dilution factor
       F = percent of positive cells / 100
       C = number of cells / well
       V = volume of viral inoculum

7. Adenoviral transduction of target cells and testing of the induced protein expression

1. Day 1: Seeding the cells
   1. Seed the target cells ensuring that they are spread evenly in the wells.
2. Day 2: Transduction of the cells
   1. Detach the target cells from one well and count them.
   2. Calculate the appropriate volume of adenoviral suspension required to transduce the cells with the desired number of infectious particles per cell.
   3. Add the corresponding amount of viral suspension to the target cells.
3. Day 3: Removal of the viral suspension and checking for GFP expression
   1. Replace the cell culture medium containing adenoviral particles with fresh medium.
   2. Check the GFP expression at the fluorescence microscope.