Editing Adenovirus pAd52 (section)

== Plaque Screening<br> ==

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.<br>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.<br><br>