Generating and Amplifying Adenoviral Constructs

Materials

• Targetting Construct in pAdtrack vector or the like (see Preparing an Adenoviral shRNA Clone )
• PmeI restriction enzyme (or other enzyme as required for linearization.
• PacI restriction enzyme
• Electrocompetent BJ5183-AD-1 cells (Agilent Cat#200157; or prepare yourself)
• 7.5 M ammonium acetate
• 20mg/mL glycogen
• 25:24:1 phenol/chloroform/isoamyl alcohol, pH 8.0
• 100% ethanol
• 2 mm electroporation cuvette
• 25 cm2 and 75 cm2 tissue culture flasks.
• Ultraclear pollyallomer tubes for Ti45 rotor (Beckman Cat# 344087)
• 2x Virus storage buffer (10 mM Tris, pH 8.0, 100 mM NaCl, 0.1% BSA and 50% glycerol; filter sterilized)

Protocol

see Nature Protocols, Current Protocols in Human Genetics protocols.

Linearization and Purification of Shuttle DNA

1. Digest 0.2-0.5 ug plasmid DNA in 100 uL volume with 30-100U of PmeI overnight at 37C. If desired check a small amount by electrophoresis to ensure complete ligation.
2. Add 100 uL ddH2O, 100 uL 7.5 M ammonium acetate and 2 uL 20 mg/ml glycogen.
3. Add 300 uL 25:24:1 phenol/chloroform/isoamyl alcohol, pH 8.0.
4. Remove top layer to clean tube and add 600 uL of 100% ethanol
5. Centrifuge 5 min at 13 000 RPM
6. Wash three times with 70% ethanol to remove residual salt.
7. Dry and resuspend in 8 uL

Transformation into BJ5183-AD-1 cells

1. Add 8 uL resuspended linearized plasmid to 20 uL electrocompetent cells
2. Carefully transfer to ice cold 2 mm electroporation cuvette avoiding bubbles and keeping on ice
3. Electroporate at 2,500 V, 200 O and 25 mF
4. Resuspend in 500 uL LB and plate on 2-5 LB/Km plates
5. Pick 10-20 of the smallest colonies the next day and grow in LB/Km broth
6. Miniprep clones. Digest 10 uL with PacI and check size by running out both digested and undigested clones on a 0.8% agarose gel. Correct recombinants usually yield a large fragment (approximately 30 kb) and a smaller fragment of 3.0 or 4.5 kb.
7. Retransform and amplify correct clone(s).

Transformation into 293A Cells

1. Plate cells so that at time of transformation they are 50-70% confluent.
2. Digest recombinant plasmid with PacI in 100 uL with 3ug plasmid and 100U PacI.
3. Precipitate with 70% ethanol, dry and resuspend in 20 uL sterile water.
4. Mix 3 ug PacI digested plasmid and 15 uL LipofectAMINE reagent for each 25-cm2 tissue culture flask in 500 uL Opti-MEM I, and incubate the DNA/LipofectAMINE reagent mix for 15–30 min at room temperature.
5. While waiting, wash cells with serum free DMEM and add OptiMEM (5 mL/10 cm dish)
6. Add DNA/Lipofectamine and incubate 4-6h.
7. Replace media with complete DMEM
8. The next day check transformation efficiency by GFP fluoresence (for pAdtrack)
9. Do not remove media, but add 2 mL fresh complete DMEM every 5-7 days. Wait 2-3 weeks before collecting viral particles.

Preparation and Purification of Viral Particles

1. Scrape cells into 15 mL conical tube.
2. Aspirate all but the final 2 mL of cells and resuspend by vortexing
3. Release viral particles with four freeze-thaw cycles (liquid nitrogen then 37C water bath)
4. Centrifuge at 500g at 4C to pellet debris.
5. Store supernatant (virus) at -80 or use immediately to amplify

Amplification of Adenovirus

1. Plate 293A cells in 25-cm2 tissue culture flasks at 80–90% confluency in 7 ml complete DMEM 6–15 h before infection.
2. Infect 293A cells by adding 40–50% of the primary transfection viral supernatants (i.e., 0.5–1.0 ml of the 2.0 ml viral lysate) to each 25-cm2 flask.
3. Check transduction by GFP fluorsesence (for pAdtrack)
4. Collect cells by scraping into a 15 mL conical 3-5 days post-infection.
5. Remove all but 5 mL media by aspiration.
6. Freeze/Thaw cells 4 times as described above
7. Centrifuge at 500g to pellet debris
8. Store supernatant (virus) at -80 or use immediately to amplify again.
9. Repeat amplification three time more first with one 75 cm2 flask (using entire supernatant) then with 3-5 75 cm2 flasks, then with 10-20 75cm2 flasks using entire viral supernatant as the innoculant each time.
10. For final amplification resuspend pelleted cells in 8 mL D-PBS -/-, perform 4 freeze thaw cycles and centrifuge 10 min at 7000g

Purification of Adenovirus

1. Transfer supernatant to 50 mL conical tube and add 4.8g of Cesium chloride and dissolve
2. Transfer 10 mL to 12 mL polyallomer tubes (for SW 45-Ti) add ~ 2mL mineral oil to top of tube to prevent tube crushing.
3. Centrifuge 18–24 h at 176,000g (SW 41 Ti rotor at 32,000 r.p.m.)
4. The purified virus should be 1-2cm below the mineral oil in an opaque layer.
5. Remove virus layer to a clean falcon tube using a syringe (do this in a beaker to easier add bleach to waste).
6. Add an equal volume of 2X Virus storage buffer and store at -80
7. Titer the virus using a kit.

desalting of CsCl purified adenovirus using PD-10 desalting columns (17-0851-01 GE Healthcare)

1. Cut column, discard storage solution and wash X5 with ~5ml PBS
2. Add virus sample (up to 2.5 ml)
3. Elute with 5 ml PBS, discard first 20 drops and collect 10 drop fractions into 10 tubes.
4. OD 260 fractions in nanodrop, virus is in middle fractions (5-7 in my extraction). Good virus yield is >3 OD.
5. Add 10% glycerol and store at -80.

Tail Vein Injection

1. Insert mouse to restriction device. Carefully warm tail with lamp/warm water, inject 0.1 OD/mouse (0.15 for HFD mouse) using insulin syringe.
2. Injected mice are housed in biohazard mouse room for 3 days (need bright-pink barcode stickers and water bottles).
3. Adenovirus is detected in liver for at least 2-3 weeks, shRNA has maximal effect at 4-6 days, usually gone in 10 days.