Adenovirus pAd52

Generation of Recombinant Adenovirus GeneExpressoMax Protocol

1. Grow 293A cells in 6 well plate  to be about 70-80% confluent at time of use (1 to 2 days) using DMEM + 10% bovine serum.

2. Add plasmid DNA (2-5 μg/per well of each plasmid) to 100 μl DMEM without serum, mix well—> Solution A.

3. Add 6 to 10 μl GeneExpressoMax to 100 μl DMEM without serum, mix well–> Solution B.

4. Mix Solution A and B, incubate at room temperature for 20 min.

5. Add mixture to adherent 293 cell cultures dropwise around the dish without removing the medium.

6. Incubate at 37oC in a CO2 incubator for 2 days.

7. Aspirate medium and overlay with 3 ml F11 + 5% Horse Serum + 0.5% agarose* or refeed with 2 ml F11 + 5% Horse Serum**.

8. Incubate at 37oC. Plaques or CPE should appear after about 5 days. Plaques can be isolated for expansion by 10 days. Note that some vectors may replicate slowly and in such cases an additional 5 ml overlay can be added to the dishes at 10 days to allow for further plaque development.

Notes
* Prepare by mixing equal volumes of DMEM + 10% Horse Serum + Pen-Strep + L-Glutamine + 0.2% Yeast Extract and 1% Agarose. (If contamination with molds is an occasional problem fungizone can be added.)
** It is good laboratory practice to use plaque purified virus prior to extensive experimentation (i.e. use an agar overlay at step 8) but recombinant virus can also be recovered by incubation of transfected cells under liquid medium. Cpe can usually be detected by 5 days and should be complete by 7-8 days.

Plaque Screening

General comments. Although 100% of viruses recovered by the Ad52 method should contain and express the desired transgene it is good laboratory practise to confirm the structure of the virus isolates to be used in large scale vector production and to prepare titered stocks of virus before embarking on extensive experimentation. Of course those who are impatient (who isn’t?) may wish to carry out preliminary experiments (expression assays using Westerns, ELISAs or IPs for example) as soon as expanded virus preparations are available, such as those obtained at step 5 below.

The protocol below is designed for 293 cells.

1. Set up 60mm dishes 293s as for plaque assays or cotransfections, i.e. to be about 80% confluent at time of use. The denser and older the cell monolayer the longer it takes for virus cpe to reach completion. Use dishes the next day or after 2 days.

2. Pick well isolated plaques by punching out agar plugs using a sterile Pasteur and transfer mashed agar to 1ml of PBS++ plus 10% glycerol. This can be stored at -70oC until results of analysis are available.

3. Remove medium from 293 dishes and add 0.2 ml virus suspension from step 2. Distribute over monolayer and adsorb at room temp for 30-60 min occasionally rocking the dishes to spread the inoculum over the cells. Add 5 ml F11 + 5%HS and incubate at 37oC.

4. Depending on size of the plaque and growth properties of the recombinant virus you should start to see cpe within 2-3 days. Do not attempt to harvest before cpe is absolutely complete, ie essentially all cells rounded and many floating (usually 3-4 days).

5. Process dishes with complete cpe as follows: leave dishes undisturbed in a laminar flow hood for 20-30 min to allow any cells in supension to settle. Gently remove medium with a pipette and save about 4ml or so in a sterile glass vial containing 0.5 ml sterile glycerol, for storage at -70oC. Remove residual medium by suction. If all this is done carefully the majority of loose cells will be left behind in the dish. The recovered medium should contain significant amounts of virus at titres at least 107 and 108 PFU/ml and can be used in preliminary experiments or for further virus expansion.

6. Add 0.5 ml pronase-SDS (see plasmid DNA purification protocol for recipe) and digest at 37oC for 3-4 hrs or overnight.

7. Transfer viscous lysate to a 1.5 ml Eppendorf tube and extract once with 0.5 ml phenol (saturated with buffer). Collect the aqueous phase with an Eppendorf pipetter and transfer to a fresh tube.

8. Add 1 ml 96% ethanol, vortex lightly or mix by tipping tube. You should get an easily visible fibrous precipitate.

Spin and wash 2x with 96% ethanol to remove phenol.

9. Dry completely in a 37oC warm room or heating block and redissolve DNA in 50ul 0.1 x SSC or 10mM Tris 1mM EDTA. It is advisable to tap the tube sharply to suspend the DNA then leave the sample overnight at 4C to allow the DNA to dissolve.

10. Digest 5-10 μl with any suitable restriction enzyme (Hind III is often the best all purpose diagnostic enzyme) for 3-4 hrs or preferably overnight. The careful worker will have already constructed the expected vector on the computer and will have created predicted maps and diagnostic restriction patterns, using, for example Vector NTi. Complete sequences for all AdMax plasmids are available from Microbix as ASCII files or as Vector NTi files.

11. Run on 1% agarose gel with an appropriate marker.
Comments: If cpe was complete you should get a relatively “pure” preparation of viral DNA (roughly 50% viral DNA – 50% cellular) with a background of cellular DNA running as a smear (some enzymes, eg Pvu I, cut human DNA infrequently in which case the cellular DNA will remain near the top of the gel). There should be very little RNA. If Hind III was used there will be a band of cellular repetitive DNA at around 1.8kb (derived from a LINE-1 element), not to be confused with viral DNA.

High Titer Adenovirus Production

General comments. Many experimental studies can be carried out using virus in the form of crude infected cell lysates. The following protocol results in high titre virus preparations. Recombinant Ads can be produced using either monolayer (see above) or suspension cultures. However, due to the greater ease of handling suspension cultures, these are preferable for the production of large amounts of high-titer viral stocks. Suspension adapted 293 cells (293N3S) are available some vendors

Protocol
1. For infection with replication-defective viruses (E1 insertion recombinants) grow 293N3S cells in spinner culture to a density of 2-4 x 105 cells/ml in 4 liters complete Joklik’s modified MEM + 10% HS. For growth of wt adenovirus or replication-proficient E1+ viruses (eg E3 insertion recombinants), grow 4 liters KB cells to a density of 5-6 x 105 cells/ml in complete Joklik’s modified MEM + 10% NBS. Centrifuge cell suspension at 750 g for 20 min, saving half of the conditioned medium. Resuspend the cell pellet in 0.1 vol fresh medium, and transfer to a sterile 500-ml bottle containing a sterile stir bar.

2. Add virus at MOI of 10-20 PFU/cell and stir gently at 37oC. After 1 hr, return the cells to the 4-liter spinner flask and bring to the original volume using 50% conditioned medium and 50% fresh medium. Continue stirring at 37oC.

3. Monitor infection twice daily by inclusion body staining as follows:

a. Remove a 5-ml aliquot from the infected spinner culture. Spin for 10 min at 750 g and resuspend the cell pellet in 0.5 ml of 1% sodium citrate.
b. Incubate at room temperature for 10 min; then add 0.5 ml Carnoy’s fixative and fix for 10 min at room temperature.
c. Add 2 ml Carnoy’s fixative, spin 10 min at 750 g, aspirate, and resuspend the pellet in a few drops of Carnoy’s fixative. Add one drop of fixed cells to a slide and air-dry for about 10 min; then add one drop orcein solution and a coverslip and examine in the microscope. Inclusion bodies appear as densely staining nuclear structures resulting from accumulation of large amounts of virus and viral products at late times in infection. A negative control should be included in initial tests.

4. When inclusion bodies are visible in 80-90% of the cells (2 to 3 days depending on the input MOI), harvest by centrifugation at 2000 g for 20 min in sterile 1-liter bottles. Combine pellets in a small volume of medium, and spin again. Resuspend pellet in 20 ml PBS++ and 10% glycerol. Freeze and thaw 2-3 times then aliquot and store at -70oC until use. Titrate by plaque assays on 293 monolayer cells.

Solutions

1. Complete Joklik’s modified MEM + 10% HS (or 10% NBS): Add 50 ml HS (or NBS) to 450 ml complete Joklik’s modified MEM (described in Section B). Store at 4oC.

2. 1% sodium citrate: Dissolve 1 g trisodium citrate dihydrate in H2O to a final volume of 100 ml.

3. Carnoy’s fixative: Add 25 ml glacial acetic acid to 75 ml methanol.

4. Orcein solution: Add 1 g orcein dye to 25 ml glacial acetic acid plus 25 ml H2O. Filter through Whatman No. 1 paper.

Production and Purification of High Titer Adenovirus Stock

General comments. Many experimental studies can be carried out using virus in the form of crude infected cell lysates prepared as described above. However, for some experiments, particularly for animal work it is usually desirable to use purified virus. The following protocol is adapted from Hitt et al. (1998).

Recombinant Ads can be purified from crude lysates of either monolayer or suspension cultures. Suspension cultures are easier to harvest, but it is easier to determine the correct time to harvest (i.e., complete or near complete cpe) when using monolayer cultures. A subline of 293 cells, 293N3S cells, adapted to growth in suspension is available from Microbix. The following protocol describes a method for banding virus obtained from 3-liters of infected suspension culture, or about 30 x 150-mm dishes.

Protocol
1. Prepare crude cell lysate from infected cells:

a) For 3-liter spinner cultures: when inclusion bodies are visible in 80-90% of the cells, harvest by centrifugation at 750 x g for 20 min in sterile 1-liter bottles. Combine pellets in a small volume of medium, and spin again.

Resuspend pellet in 15-30 ml 0.1M Tris-HCl, pH 8.0: if wild-type levels of virus are expected, resuspend in 30 ml, if lower yields are expected (some vectors may replicate to lower yields than others), use 15 ml. Store sample at -70oC.

b) For 30 x 150-mm dishes: when all infected cells are rounded and most are floating, scrape dishes, then transfer cells and supernatant to 50-ml centrifuge tubes and spin 10 min at 750 x g. Resuspend cell pellet in 15 ml 0.1M Tris-Cl, pH 8.0. Store sample at -70oC.

2. Thaw sample and add 1.5 ml 5% Na deoxycholate for each 15 ml of cell lysate. Mix well and incubate at room temperature for 30 min. This results in a relatively clear, highly viscous suspension.

3. Add 150 μl 2 M MgCl2 and 75 μl DNase I solution for each 15 ml of cell lysate, then mix well. Incubate at 37oC for 30-60 min, mixing every 10 min. The viscosity should be reduced to only slightly more than that of water.

4. Spin 15 min, top speed, at 5oC in the Beckman table top centrifuge.

5. Meanwhile, prepare CsCl step gradients (one SW41 ultraclear tube for each 5 ml of sample) in the virus hood: Add 0.5 ml of 1.5g/cc solution to each tube. Gently overlay with 3.0 ml of 1.35g/cc solution. Gently overlay this with 3.0 ml of 1.25g/cc solution. Do not disturb gradients once they are formed.

6. Apply 5 ml of supernatant from step 4 to the top of each gradient. If necessary, top up tubes with 0.1M Tris-Cl, pH 8.

7. Spin at 35K rpm in SW41 rotor, 10 oC, for 1 hour (accelerate/decelerate set to 1).

8. Collect the viral bands (should be at the 1.25g/cc-1.35g/cc interface) and pool. (One can collect by sealing the top of the tube with a piece of parafilm, puncturing the parafilm with a needle, then puncturing the bottom of the tube with a red hot 16 gauge needle, controlling the flow of solution out the bottom with a gloved finger over the top hole.)

9. Transfer the pooled virus to a SW50.1 ultraclear tube (using the 1.35g/cc CsCl solution to top up the tube), mix well, and centrifuge in the SW50.1 rotor at 35K rpm, 4oC, for 16-20 hr. (Alternatively, the pooled virus can be centrifuged in the SW41 rotor at 35K rpm, 10oC, 16-24 hr.)

10. Collect the virus band in the smallest volume possible (usually 0.5 to 1 ml), transfer to a Slide-A-Lyzer dialysis cassette, and dialyze at 4oC against 3 changes of 500 vol (or more) 10 mM Tris-HCl, pH 8.0, for at least 24 hr total.

11. After dialysis, add sterile glycerol to a final concentration of 10%. Store the purified virus at -70oC in small aliquots.

Materials

The following special equipment is required:
Beckman SW 41 rotor,
SW50.1 rotor, ultraclear tubes for each rotor,
Pierce Slide-A-Lyzer dialysis cassettes, sterile 1-liter beaker with stir bar, syringes and needles.

Solutions
1. 0.1 M Tris-HCl, pH 8.0: Add 1.2 g Tris base to 80 ml H2O. Adjust pH to 8.0 with HCl. Adjust volume to 100 ml and autoclave.

2. 5% Na deoxycholate: Add 5 g Na deoxycholate to 100 ml H2O.

3. 2M MgCl2: Add 40.6 g MgCl2.6H2O to 100 ml H2O and sterile filter.

4. DNase I solution: Dissolve 100 mg bovine pancreatic deoxyribonuclease I (DNase I) in 10 ml of 20 mM Tris-HCl, pH 7.4, 50 mM NaCl, 1 mM dithiothreitol, 0.1 mg/ml bovine serum albumin, and 50% glycerol. Store in small aliquots at -20oC.

5. CsCl solutions for banding:
density CsCl 10 mM Tris, pH8
1.50 g/cc 90.8 g 109.2 g
1.35 g/cc 70.4 g 129.6 g
1.25 g/cc 54.0 g 146.0 g

Add 10 mM Tris to indicated amount of CsCl and stir to dissolve. Verify density by weighing 1 ml of each solution. Sterile filter and store at room temperature.

6. Dialysis buffer: 10 mM Tris, pH 8.0. Use at 4oC.

7. Sterile glycerol: Prepare by autoclaving.