Adenovirus AdEasy

Generation of Recombinants in Bacterial Cells

1. Prepare electrocompetent BJ5183 cells in 20 ml/tube aliquotes.

2. Linearize the shuttle plasmids with PmeI. Usually one-fifth of a miniprep (typically 100-500 ng) is sufficient. After digestion, DNAs are phenol-chloroform extracted, ethanol precipitated, and resuspended in 6.0 ml H2O.

3. Co-transform PmeI-digested shuttle plasmid with 1.0 ml adenoviral backbone vector (100 ng/ul). Twenty ul of electrocompetent E. coli BJ5183 cells were added and electroporation was performed in 2.0 mm cuvettes at 2,500V, 200 Ohms, and 25 mFD in a Bio-Rad Gene Pulser electroporator. Resuspend transformation mix in 500 ml L-broth (incubation at 37oC for 10-20 min is optional, depending on background, see FAQ's). Plate on 4 to 5 LB/kan plates, and grow at 37oC overnight (16-20 hrs).

4. Pick up 10 to 20 smallest colonies, and grow them in 2 ml L-broth containing 25 mg/ml kanamycin for 10-15 hours.

5. Perform minipreps using the conventional alkaline lysis method, and check the sizes of supercoiled plasmids by running one-fifth of a miniprep on 0.8% agarose gel.

6. Restriciont digest DNA from clones with PacI. Candidate clones usually yield a large fragment (near 30 kb), plus a smaller fragment of 3.0kb or 4.5kb.

7. Re-transform one microliter of the correct recombinant plasmid minipreps into DH10B (or your favorite plasmid propagation strain). Plasmids are purified by CsCl -banding (see Appendix D) or using commercially available purification kits for transfection of 293 cells.

Viral Production in 293 or 911 Cells

1. 293 cells (E1-transformed human embryonic kidney cells) or 911 cells (E1-transformed human embryonic retinal cells) in one or two T-25 flasks at 2 x 106 cells per flask ~24 hours prior to transfection. The confluency should be about 50% to 70% at the time of transfection.

2. On the day of transfection, digest recommbinant adenovial plasmids with PacI (usually 4 µg DNA is needed to transfect one T-25 flask). Ethanol precipitate the plasmids and resuspend in 20 µl of sterile H2O.

3. Perform a  transfection using GeneExpressoMax. Mix 4 µg of PacI-digested plasmid and 12 µl of GeneExpressoMax for each T-25 in 500 µl of OptiMem I or DMEM medium, and incubate at room temperature for 20 min.

4.  Add GeneExpressoMax-DNA mix to the flasks, and return to 37oC CO2 incubator.

6. Remove medium containing GeneExpressoMax-DNA mix 48 hours later, and add 6 ml fresh complete DMEM (10% FBS, 1% Pen/Strep). Do not change the GeneExpressoMax/DNA medium if a significant amount of floating cells are observed; sometimes this happens with 293 cells and doesn’t necessarily indicate a problem. If lots of floating cells are seen, add 6.0 ml complete DMEM to each flask and incubate at 37°C overnight. Change medium next morning.

7. Transfections and viral productions can minotored by GFP expression if pAdTrack-based vectors are used. No obvious plaques or CPE are easily observed by standard microscopy up to 2 weeks post transfection in most cases. However, plaques are always observed under fluorescence using the GFP marker.

8. Scrape cells off flasks with a rubber policeman (not trypsin) at 7 to 10 days post-transfection and transfer to 50 ml conical tubes. Spin cells in a benchtop centrifuge and resuspend pellet in 2.0 ml HBSS or sterile PBS. Freeze cells in dry ice/methanol bath, and thaw in a 37oC water bath. Vortex vigorously. Repeat freeze/thaw/vortex for 3 more cycles (four cycles total). Do not let virus supernatants warm up. Spin samples briefly and store supernatant at -20oC.

9. Infect two 50% to 70% confleunt T-25 flasks of 911 or 293 cells using 30-50% of the above viral supernatants for each flask. CPE or cell lysis should become evident at 2 to 3 days post infection. Productive infections easily observed with the AdTrack vectors.

10. Collect viruses when a third to half of the cells are detached, usually 3 to 5 days post infection. Presence of the recombinant adenoviruses can be confirmed by Western blot and/or PCR (for PCR, take 5 µl virus sup plus 10 µl PCR-grade Protease K at 55oC for 1 hour, then boil samples for 5 min. spin briefly, use 1 to 2 µl for PCR).

11. Scrape cells off and prepare viral supernatants as described in Step 8 above. You should have at least 107 infectious particles/ml at this stage, and often much more. Each round of amplification should give at least 10-fold more virus than present in the previous round.

12. To amplify further, repeat the infection of cells using 30-50% of the viral supernatant from Step 11, using T75 flasks instead of T25 flasks. Titers can be measured at any time, which is particularly easy with AD-Track vectors. Simply infect 293 cells with various dilutions of viral supernatant and see how many are green 18 hours later. Without AdTrack, viruses can be plaque titered or titered by limiting dilution using standard methods; we find these methods much less simple and quantitative than that employing the GFP marker, but these have to be used if GFP is not present.

Preparation of High Titer Viral Stocks

1. Plate 911 or 293 cells in T-75 flasks to be 90% confluent at time of infection (about 1x107 cells/T-75). Usually, fiften to twenty T-75 flasks are sufficient to make a high titer stock.

2. Infect cells with virus sup at a multiplicity of infection (MOI) of 5 to 10 PFU per cell. When all cells have rounded up and about half of the cells are detatched (usually at 3 to 4 days post infection), harvest and combine all flasks. Spin 5 min in a benchtop centrifuge (~500 g), and remove the supernatant.

3. Resupend pellet in 8.0 ml sterile PBS. Perform four cycles of freeze/thaw/vortex. Centrifuge lysate in Sorvall HS4 rotor at 6000 rpm (7000 g) 4oC for 5 min.

4. Weigh 4.4 grams of CsCl in a 50-ml conical tube, transfer 8.0 mls of clear virus sup to the tube, and mix well by vortexing. Transfer the CsCl solution (about 10 ml, density of 1.35 g/ml) to a 12 ml polyallomer tube for SW41 rotor. Overlay with 2 ml mineral oil. Prepare a balance tube. Spin the gradient in SW41 rotor at 32,000 rpm, 10oC, for 18 to 24 hours.

5. Collect virus fraction (about 0.5 to 1.0 ml) with a 3cc syringe and an 18g needle. Mix with equal volume 2X Storage Buffer (2X Storage Buffer = 10 mM Tris, pH 8.0, 100 mM NaCl, 0.1% BSA, and 50% glycerol, filter sterilized). Store virus stocks at -20oC.

6. Check viral titer by GFP (preferred) of by plaque assays (see below) or by immunohistochemical staining, or simply read OD at 260 nm. To read OD, add 15 µl virus to 15 µl blank solution (Blank Solution = 1.35g/ml CsCl mixed with equal vol 2X Storage Buffer) plus 100 µl TE/0.1% SDS; vortex 30 seconds, centrifuge 5 min. measure A260. One A260 unit contains ~1012 viral particles (particles:infectious particles =~20:1).

Plaque Assay for Purification and Titration of rAdenoviruses

Note:  this only needs to be done for adenoviruses without GFP marker.  In those which include GFPmarker, titration of infectious units (i.e., those resulting in expression of GFP) is simply determined by fluorescence microscopy of your favorite cell type.

1. Remove all but 2 ml per well of medium from  6-well plates containing 80% to 90% confluent 911 or 293 cells.  Infect with appropriately diluted virus for 3 to 6 hours.  Infect cells with 6 different dilution titers (e.g. 10^-3 to 10^-8ml/well).

2. Prepare the overlay agar as following: autoclave 100 ml of 2.8% Bacto-Agar (Difco) and keep warm at 45^oC water bath. For 100 ml overlay:

	Stock Conc	Final Conc.	ml/100 ml overlay
2 X BME (GIBCO)	2 X	1X	50.0
HEPES	1.0M	20.0mM	2.0
MgCl2	1.0M	12.5mM	1.25
FBS	100.0%	10.0%	10.0
Pen/Strep	100 X	1 X	1.0
mix well and incubate at 37oC bath.
Bacto-Agar	2.8%	1.0%	36.0

Mix well and swirl at 37^oC water bath, add 4 ml/well for 6-well plate.

Note:  If you want to melt 2.8% agar stock,  microwave or heat it in boiling water bath.  Prepare 50 ml overlay at a time. To prevent the pre-drying of agar before plaque formation, it is important to add sterile PBS or Hank’s medium in the space between wells, and wrap the plates with SaraWrap.

3. Return the plates to 37^oC CO₂ incubator.  On days 5 - 7, overlay 2 to 3 ml agar containing nuetral red (from 100x stock, available from GIBCO-BRL) to each well.  Plaques should be visible 16-30hrs after the neutral red overlay. (In general, plaques should form in 911 cells in 4 or 5 days and in 293 cells after 5-7 days).

4. Pick 5 to 10 well-isolated plaques for each recombinant by punching out agar plugs with a sterile pasteur pipet. Store agar plugs in 200µl HBSS medium. Perform freeze/thaw/vortex cycle 4 times. Spin samples briefly.

5. Infect one well of 6-well plate of 911 or 293 cells (about 80-90% confluency at time of infection) with 50-70% virus sup of each plaque for 3 to 5 days (depending on the titer of individual plaque). 

Presence of recombinant virus can be confirmed by Western blot or PCR.

6. Collect viral supernatant using four cycles of freezing/thawing, as described earlier in this guide. The virus sup can be used to further infect T-25, and then T-75 flasks, for large scale preparation.