Construction and Characterization of Adenovirus Vectors

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Figure 1. Schematic representation of the AdEasy system for Ad vector production. See Discussion for details. (Modified from He et al. 1998 , with permission from National Academy of Sciences © 1998.)

MATERIALS

Reagents

Agar

Agarose (ultrapure, 1% w/v)

Sterilize by autoclaving for 20 min at 121°C. Store at room temperature and melt in a microwave oven before use.

Agarose gel (0.8%) and reagents for agarose gel electrophoresis

Bacteria: BJ5183 cells, RecA -proficient

These cells are supplied in a number of commercially available AdEasy kits.

Bacteria: DH5 , RecA -deficient

Prepare transformation-competent cells with rubidium chloride and store in 0.2-mL aliquots at -80°C.

Cells: A549

Cells: 293, low-passage

Grow 293 and A549 cells in 150-mm dishes in a 37°C, 5% CO₂ incubator and split 1:2 or 1:3 when they reach ~90% confluence.

Cells: 293N3S

Maintain 293N3S cells in 150-mm dishes as described above or in suspension. Grow the cells in suspension in 1X maintenance medium in suspension flasks, agitated at 70 rpm. When they reach a density of 5 x 10⁵ cells/mL, dilute 1:2 or 1:3 with 1X maintenance medium.

CsCl (cesium chloride), 1.25 g/mL and 1.35 g/mL, in dialysis buffer

Prepare each gradient solution separately. Sterilize by filtration through a 0.2-µm filter.

Chloroform:isoamyl alcohol (24:1)

Citric saline (2X)

For 1X citric saline, dilute the 2X stock solution with H₂ O and sterilize by autoclaving for 45 min at 121°C.

Complete medium

Dialysis buffer (10 mM Tris-HCl, pH 8.0)

Sterilize by filtration through a 0.2-µm filter.

DNase I (Ad)

Ethidium bromide

Isopropanol

Kanamycin

LB (Luria-Bertani) liquid medium

Maintenance medium (1X)

Maintenance medium (2X)

MgCl₂ (2 M)

Sterilize by autoclaving for 45 min at 121°C.

Minimum essential medium (MEM)

NaCl (5 M)

Sterilize by autoclaving for 45 min at 121°C.

PBS-Ad

Phenol (buffer-saturated)

Plasmids (shuttle and backbone)

These are supplied in a number of commercially available AdEasy kits.

Restriction endonucleases: PacI, PmeI

Other enzymes may be needed for cloning the gene of interest into the shuttle vector.

RNase A (10 mg/mL)

SDS (sodium dodecyl sulfate) (0.1% w/v, in TE)

SDS-proteinase K solution

Sodium deoxycholate (5% w/v)

Sterilize by filtration through a 0.2-µm filter.

Sucrose (40% w/v, in PBS-Ad

Sterilize by filtration through a 0.2-µm filter. For 4% sucrose, dilute the stock solution with PBS-Ad and sterilize by filtration before use.

TE-Ad (1X)

For 0.1X TE-Ad, dilute the 1X stock solution with H₂ O and then sterilize by autoclaving for 45 min at 121°C.

Transfection reagent (e.g., SuperFect; QIAGEN)

Trypsin-EDTA (10X) (Invitrogen 15400054)

Dilute the 10X stock solution with PBS-Ad to 1X before use.

Equipment

Agarose gel electrophoresis equipment

Bottles (polypropylene, 500- and 1000-mL)

Centrifuge (high-performance) (e.g., Avanti J-series, Beckman)

Cryovials (4-mL)

Dialysis cassettes (Slide-A-Lyzer, 10,000-kDa molecular mass cutoff, 0.5-3.0 mL) (Pierce 66380)

Dishes (six-well)

Flasks (spinner, with impeller assembly, 250- and 3000-mL) (Bellco Glass 1965-61002 and 1965-61030, respectively)

Heat sealer

Hemacytometer

Hood (laminar flow)

Ice

Incubator preset to 37°C, 5% CO₂

Microcentrifuge

Needle (22-gauge)

Pasteur pipettes (sterile, cotton-plugged)

Petri dishes (35-, 60-, and 150-mm)

Pipettes

Rotor (70.1 Ti) (Beckman)

Rotor (JLA 10.500) (Beckman)

Rotor (swinging bucket, SW 41 Ti) (Beckman)

Shaker (platform)

Spectrophotometer

Stirrer (magnetic)

Syringe (3-cm³ )

Tubes (conical, 50-mL)

Tubes (microcentrifuge, 1.5-mL)

Tubes (polypropylene, capped, 13-mL)

Tubes (polystyrene, conical, 15-mL)

Tubes (polystyrene, round-bottom, 5-mL)

Tubes (ultracentrifuge, Quick-Seal, 16- x 76-mm) (Beckman 344322)

Tubes (ultracentrifuge, 14- x 89-mm)

Ultracentrifuge

Vortexer

Water baths preset to 22°C, 37°C, 42°C, and 56°C

METHOD

Generation of Infectious Ad Plasmids

1. Generate a shuttle vector containing the transgene of interest using standard cloning procedures.
 

2. Digest 2 µg of the shuttle vector with 1 µL of PmeI in a total volume of 20 µL overnight at 37°C.
 

3. Add 3 µL of PmeI-digested shuttle and 3.3 µL of 0.1 µg/µL supercoiled pAdEasy to a 13-mL capped polypropylene tube. Chill on ice.
Add 3 µL of PmeI-digested shuttle without pAdEasy to another tube as a negative control.
 

4. Thaw two 0.2-mL aliquots of BJ5183 competent cells on ice.
 

5. Add 0.2 mL of BJ5183 cells to the DNA. Incubate for 25 min on ice.
 

6. Incubate the tubes for 90 sec at 42°C and then for 2 min on ice.
 

7. To each tube, add 1 mL of LB. Incubate with shaking at 225 rpm for 25 min at 37°C.
 

8. Transfer the bacteria to 1.5-mL tubes. Centrifuge at 8500g for 1 min at room temperature.
 

9. Aspirate 1 mL of LB. Resuspend the pellet in a total volume of 0.2 mL.
 

10. Spread 0.1 mL of bacterial suspension on the surface of two Petri dishes containing 1.5% solid agar in LB supplemented with 25 µg/mL kanamycin. Use one plate for the control suspension.
No colonies should form on the negative control plate. However, if "background" colonies do appear, recombinant colonies will be noticeably smaller than those produced by bacteria transformed with undigested shuttle plasmid.
See Troubleshooting.
 

11. Purify the plasmid DNA from four to eight small colonies by small-scale alkali lysis (see Preparation of Plasmid DNA by Alkaline Lysis with SDS: Minipreparation (Sambrook and Russell 2006 ). Suspend the DNA in a total volume of 25 µL of 0.1X TE-Ad.
 

12. Repeat Steps 5-10 with 5 µL of DNA (from Step 11) and 100 µL of DH5 cells, rather than BJ5183.
 

13. Select two colonies from each plate. Purify the DNA by small-scale alkali lysis, as above.
Digest the DNA with several different restriction enzymes to verify the structure of the plasmid. Correct clones should by subjected to large-scale purification by CsCl buoyant density centrifugation and then used for generation of a recombinant Ad vector (see Step 15).

^{Cell Preparation}

^{14. Prepare the cultured cells for transfection.}

^{To prepare adherent 293 and A549 cells:}

^{i. Remove medium from 150-mm dishes of 293 or A549 cells.}

^{ii. Rinse the monolayer twice with 5 mL of 1X citric saline (for 293 cells) or 2 mL of 1X trypsin-EDTA (for A549 cells).}

^{iii. For 293 cells, remove all but 0.5 mL of the citric saline after the second rinse and leave the dishes for 5-10 min at 22°C until cells begin to detach. For A549 cells, add 2 mL of trypsin-EDTA and leave the dishes for 5 min at 37°C until the cells begin to detach. Tap the sides of the dishes to detach all cells.}

^{iv. Dilute the cells to 12 mL with complete medium and distribute into new 35-mm dishes.
Incubate the plates so that the cells are ~90% confluent on the day of transfection.}

^{To prepare 293N3S cells in suspension:}

^{v. Transfer six 150-mm plates of confluent 293N3S cells to a 3-L spinner flask. Bring the total volume in the flask to 1 L with 1X maintenance medium.}

^{vi. Every 1-2 d, remove 2 mL of suspension cells to a 15-mL polystyrene conical tube. Add 2 mL of 2X citric saline. Vortex vigorously for 10 sec.}

^{vii. Incubate the cells for 15 min at 37°C. Vortex vigorously for 10 sec.}

^{viii. Count cells using a hemacytometer. When the cell density is >4 x 105 cells/mL, add 1 L of 1X maintenance medium until 3 L of cells are obtained with a final density of 3 x 105 to 5 x 105 cells/mL.
If cells are in clusters too large to count, continue to incubate at 37°C and vortex until the clusters are broken up.}

^{Transfection for Rescue of Ad Vectors}

^{15. Digest 10 µg of the plasmid encoding the recombinant Ad genome (obtained from Step 13) with 2 µL of PacI in a total volume of 50 µL overnight at 37°C.}

^{16. Mix 40 µL of PacI-digested DNA with 360 µL of MEM and 16 µL of transfection reagent in a 5-mL tube. Vortex vigorously for 10 sec.}

^{17. Allow complexes to form for 15 min at room temperature.}

^{18. Add 2.4 mL of MEM to each tube containing the DNA complexes. Mix well by gentle pipeting.}

^{19. Rinse four 35-mm plates seeded with 293 cells (as described in Step 14) twice with 2 mL of PBS-Ad.}

^{20. Transfer 0.7 mL of the plasmid complex suspension to each 35-mm dish of 293 cells.}

^{21. Incubate the cells for 3 h at 37°C, 5% CO₂ .}

^{22. While the cells are incubating, prepare the agarose overlay:}

^{i. About half an hour before the end of the incubation, melt the 1% agarose (minimum 1.5 mL per 35-mm dish) in a microwave oven. Equilibrate to 42°C in a water bath.}

^{ii. Equilibrate 2X maintenance medium (1.5 mL per 35-mm dish) to 37°C.}

^{23. After 3 h, remove the transfectant from the cells. Rinse the monolayer with 1 mL of PBS-Ad.}

^{24. Mix equal volumes of the agarose solution and 2X maintenance medium. Add 3 mL to each 35-mm dish of 293 cells.
This must be performed quickly to avoid solidification of the agarose, but gently to avoid disturbing the monolayer.}

^{25. Allow the overlay to solidify (~15 min at 22°C).}

^{26. Return the cells to the incubator. Incubate until plaques form (~7-12 d).
See Troubleshooting.}

^{27. Choose several well-isolated plaques. Use a sterile cotton-plugged Pasteur pipette to remove a plug of agarose over each plaque. Place each plug in a cryovial containing 1 mL of 4% sucrose. Vortex briefly. Store plaques at -80°C.
Initial Ad plaque isolates should be plaque-purified a second time (Steps 22-27) to ensure that the resulting virus is from a single clone.}

^{Analysis and Expansion of Plaque-Isolated Ad Vector}

^{28. For each sample of plaque-purified vector obtained above, add 100 µL to two 35-mm plates of 293 cells (~90% confluence).}

^{29. Return the cells to the incubator. Allow the virus to adsorb for 1 h, rocking the dishes every 10-15 min.}

^{30. After the adsorption period, add 2 mL of 1X maintenance medium to each plate. Return them to the incubator.}

^{31. Examine plates for cytopathic effect (CPE).
The cells should have a rounded morphology or be detached from the plate.}

^{32. Use one plate for analysis of the recombinant Ad structure. Reserve the contents of the other plate for vector expansion.}

^{To analyze the plaque-isolated Ad vector:}

^{i. Once complete CPE is achieved, leave the plates undisturbed for ~10 min in a laminar flow hood so that detached cells will come to rest on the bottom of the plate.}

^{ii. Carefully remove the medium. Resuspend the remaining cells in 0.2 mL of SDS-proteinase K solution. Transfer the suspension to a microcentrifuge tube.}

^{iii. Incubate the lysate overnight at 37°C.}

^{iv. Add 0.3 mL of 1X TE-Ad to the lysate. Extract with 0.5 mL of buffer-saturated phenol, followed by 0.5 mL of chloroform:isoamyl alcohol.}

^{v. Add 0.1 mL of 5 M NaCl and 0.5 mL of isopropanol.}

^{vi. Pellet the DNA by centrifugation at 20,000g for 10 min at 4°C.}

^{vii. Resuspend the DNA in 20-50 µL of 1X TE-Ad. Use 5-10 µL for digestion with appropriate restriction enzymes.}

^{viii. Examine the resulting banding pattern by electrophoresis on a 0.8% agarose gel, followed by staining with ethidium bromide.}

^{To expand plaque-isolated Ad vector:}

^{ix. Once complete CPE is achieved (see Step 31), scrape the cells from the dish into the medium. Transfer to a 4-mL cryovial.}

^{x. Add 40% sucrose in PBAd to a final concentration of 4%. Vortex briefly.
Use immediately or store at -80°C until use.}

^{xi. Infect a 150-mm dish of 293 cells with 1 mL of inoculum. Return cells to the incubator.}

^{xii. Allow the virus to adsorb for 1 h, rocking the dishes every 10-15 min.}

^{xiii. After the adsorption period, add 20 mL of 1X maintenance medium to each plate. Return them to the incubator.}

^{xiv. Examine the plates for CPE. Once complete CPE is evident, remove the cells and medium to a 50-mL conical tube. Add 1/10 volume of 40% sucrose (to a final concentration of 4% sucrose). Store inoculum at -80°C.}

^{Large-Scale Preparation of Ad Vectors}

^{33. Thaw the inoculum in a 22°C water bath. If necessary, increase the volume of the inoculum to 30 mL using MEM.}

^{For large-scale preparation of Ad vectors using adherent 293 cells:}

^{i. Working in batches of 10 plates, remove the medium from 30 150-mm dishes of 293 cells (~90% confluent at the time of infection). Replace with 1 mL of inoculum per plate.}

^{ii. Allow the virus to adsorb in a 5% CO2 incubator for 1 h at 37°C, rocking the plates every 10-15 min.}

^{iii. Add ~20 mL of 1X maintenance medium to each dish. Return the cells to the incubator. Examine the cells daily until complete CPE is evident (~2-3 d).}

^{iv. Remove the medium and cells to two 500-mL polypropylene bottles.
Most of the cells should be detached in the medium; however, any remaining cells are usually loosely attached and can be removed by tapping the sides of the dish.}

^{v. Use the same pipette to rinse groups of 10 dishes twice with 10 mL of PBS-Ad.}

^{vi. Centrifuge cells at 650g for 20 min at 4°C. Decant the medium and retain the cell pellets.}

^{vii. Resuspend the cells in 3 mL of 4% sucrose. Transfer to a 50-mL conical tube.}

^{viii. Using the same pipette, rinse the bottles once with 2 mL of 4% sucrose and once with 4-5 mL of 4% sucrose.
The total volume of the cell pellet should be ~15 mL. The cells can be processed immediately for vector purification or stored at -80°C.}

^{For large-scale preparation of Ad vectors using suspension-adapted 293N3S cells:}

^{ix. Distribute a 3-L culture of 293N3S cells into eight 500-mL centrifuge bottles.}

^{x. Centrifuge at 650g for 20 min at room temperature. Decant the medium into sterile 1-L bottles.
Return 1 L of spent medium to the 3-L flask. Return the flask to the incubator.}

^{xi. Use the spent medium to resuspend the cell pellets to a final volume of ~40 mL. Transfer the suspension to a 250-mL spinner flask. Rinse the bottles twice with 10 mL of spent medium and transfer to the spinner flask.}

^{xii. Add the thawed inoculum to the spinner flask.
The total volume in the flask should be ~100 mL.}

^{xiii. Transfer the flask to the incubator. Agitate the cells at 70 rpm for 2 h.}

^{xiv. Transfer the cells to the 3-L suspension flask containing 1 L of spent medium. Rinse the 250-mL spinner flask twice with ~250 mL of fresh 1X maintenance medium and transfer to the 3-L spinner flask. Add 500 mL of fresh 1X maintenance medium to a final volume of 2 L.}

^{xv. Remove 2 mL of the cell suspension to a 35-mm plate. Return the suspension flask and the plate to the incubator.
The cells should reattach to the plate.}

^{xvi. When complete CPE is evident on the 35-mm plate (~2-3 d), decant the suspension culture into 500-mL centrifuge bottles.}

^{xvii. Centrifuge the cells at 650g for 20 min at 4°C. Decant the medium and retain the cell pellets.}

^{xviii. Resuspend the cells in 3 mL of 4% sucrose. Transfer to a 50-mL conical tube.}

^{xix. Using the same pipette, rinse the bottles once with 2 mL of 4% sucrose and once with 4-5 mL of 4% sucrose.
The total volume of the cell pellet should be ~15 mL. The cells can be processed immediately for vector purification or stored at -80°C.}

^{Vector Purification}

^{All volumes stated below are for a cell pellet with a total volume of 15 mL. Scale volumes accordingly.}

^{34. Add 1.5 mL of 5% sodium deoxycholate to a 15-mL aliquot of cells prepared by either large-scale method (Steps 33.i-33.viii or Steps 33.ix-33.xix).
If using stored pellets, thaw in a 37°C water bath.}

^{35. Incubate with frequent inversion for 30 min at 22°C.
The lysate should have a thick, highly viscous consistency.}

^{36. Add 0.3 mL of 2 M MgCl₂ , 0.15 mL of 10 mg/mL RNase A, and 0.15 mL of 10 mg/mL DNase I (Ad). Incubate with occasional inversion for 30-60 min at 37°C.}

^{37. Once the viscosity of the lysate is near that of water, centrifuge at 1000g for 10 min at 22°C.}

^{38. Prepare CsCl step gradients in Ultra-Clear ultracentrifuge tubes (two tubes per virus).}

^{i. Add 2 mL of 1.35 g/mL CsCl to each tube.}

^{ii. Carefully (i.e., with a steady stream, at a rate of ~30 sec/mL) overlay with 3 mL of 1.25 g/mL CsCl.}

^{iii. Carefully add equal volumes of cleared lysate (~6.5-7 mL) to each tube. Balance the tubes.}

^{39. Place the tubes in the buckets of a SW 41 rotor. Use slow acceleration and deceleration profiles (500 rpm over ~5 min) to centrifuge the samples at 35,000 rpm for 1 h at 10°C.
The viral band is the lowest band visible on the gradient and will be found at the interface between the 1.25- and 1.35-g/mL layers of the gradient.}

^{40. Use a 22-gauge needle attached to a 3-cc syringe to pierce the tube ~1 cm below the virus band. Turn the bevel so that it is parallel to the band and slowly aspirate the band, lowering the needle as the band lowers.
Virus from both step gradient tubes can be combined in a single Quick-Seal ultracentrifuge tube.
See Troubleshooting.}

^{41. Fill the Quick-Seal tube containing the virus to the base of the neck with 1.35 g/mL CsCl. Use a heat sealer to seal the Quick-Seal tubes.
Prepare a balance tube by filling another Quick-Seal tube with 1.35 g/mL CsCl.}

^{42. Centrifuge at 35,000 rpm with maximal acceleration and deceleration in a 70.1 Ti rotor overnight at 10°C.}

^{43. Pierce the top of the sealed 70.1 Ti tube to form an air inlet. Use a 22-gauge needle attached to a 3-cc syringe to pierce the tube ~1 cm below the virus band. Turn the bevel so that it is parallel to the band and slowly aspirate the band, lowering the needle as the band lowers.
Take care to minimize the volume extracted.}

^{44. Inject the Ad into a prepared dialysis cassette. Remove the air bubble with the syringe.}

^{45. Dialyze the Ad vector for 24 h at 4°C against two 500-mL volumes of dialysis buffer.}

^{46. Remove the vector from the dialysis cassette. Retain the syringe and 0.9 mL of dialysis buffer.}

^{47. Add 40% sucrose to the vector and the dialysis buffer to a final concentration of 4% sucrose. Store purified vector in small aliquots (~100-200 µL) at -80°C. Store the buffer at -20°C.
Ad vector stocks are stable for years at -80°C.}

^{Characterization of Purified Ad Vectors}

^{48. Further characterize the Ad vectors by assessing the genetic structure, determining titer, and examining for contamination with replication-competent adenovirus (RCA).}

^{To confirm the genomic structure of the purified Ad vector:}

^{i. Rinse the syringe used to remove the vector from the dialysis cassette (from Step 46) with 0.2 mL of SDS-proteinase K. Transfer the liquid into a 1.5-mL tube.}

^{ii. Incubate overnight at 37°C.}

^{iii. Add 0.3 mL of 1X TE-Ad to the lysate. Extract with 0.5 mL of buffer-saturated phenol, followed by 0.5 mL of chloroform:isoamyl alcohol.}

^{iv. Add 0.1 mL of 5 M NaCl and 0.5 mL of isopropanol. Pellet the DNA by centrifugation at 20,000g for 10 min at 4°C.}

^{v. Resuspend the DNA in 20-50 µL of 1X TE-Ad. Use 5-10 µL for digestion with appropriate restriction enzymes. Examine the resulting banding pattern by electrophoresis on a 0.8% agarose gel, followed by staining with ethidium bromide.}

^{To determine titer in infectious units by plaque-forming unit (pfu) assay:}

^{vi. Prepare serial dilutions (10-4 - 10-9 ) of Ad vector in MEM.}

^{vii. Infect 293 cells (~90% confluent) in the wells of a six-well dish with 0.1-mL aliquots of each dilution. Return cells to the incubator. Allow the virus to adsorb for 1 h, rocking the dishes every 10-15 min.}

^{viii. After the adsorption period, overlay with agarose (see Steps 22-25).}

^{ix. Incubate for 10-12 d. Count the number of plaques. Multiply the number of plaques in a well by the dilution factor to determine the vector titer (pfu/mL).}

^{To determine vector titer in particles/milliliter:}

^{x. Dilute 20-50 µL of purified vector to a final volume of 1 mL in 0.1% SDS.
Use the dialysis buffer (from Step 46) as a blank control.}

^{xi. Incubate for 10 min at 56°C. Vortex briefly, and centrifuge briefly.}

^{xii. Determine the OD₂₆₀ . Using an extinction coefficient of 1.1 x 1012 for wild-type Ad (Maizel et al. 1968 ), calculate the number of particles/milliliter: (OD₂₆₀ )(dilution factor)(1.1 x 1012 ).
A typical Ad vector preparation should have a ratio of particle to pfu of ~10.}

^{To detect the presence of RCA in purified vector preparations:}

^{Recombination between vector DNA and Ad5 DNA present in 293 or 911 cells can result in transfer of the early gene E1 to the vector, generating RCA. A549 cells do not express E1 and cannot support efficient replication of E1-deleted vectors. Therefore, after infection with purified Ad, only contaminating RCA will induce CPE in A549 cells.}

^{xiii. Infect one 60-mm dish of A549 cells (~90% confluence) with 106 pfu in 250 µL of MEM. Infect a second 60-mm dish with 107 pfu in 250 µL of MEM. Infect a 150-mm dish with 108 pfu in 1 mL of MEM. Return cells to the incubator.}

^{xiv. Allow the virus to adsorb for 1 h, rocking the dishes every 10-15 min.}

^{xv. After the adsorption period, add 5 mL (for 60-mm plates) or 20 mL (for 150-mm plates) of 1X maintenance medium to each plate and return them to the incubator.}

^{xvi. Once complete CPE is evident (or after 7 d), harvest the monolayer by scraping the cells into the medium. Add 40% sucrose (to a final concentration of 4%).
Viruses can be stored at -80°C.}

^{xvii. Use 1 mL of each viral culture (from Step 48.xvi) to infect an individual 150-mm dish of A549 cells. Add 1 mL of MEM to a fourth plate as a negative control.}

^{xviii. Allow the virus to adsorb for 1 h, rocking the dishes every 10-15 min.}

^{xix. After the adsorption period, add 20 mL of 1X maintenance medium to each plate. Return them to the incubator.}

^{xx. Compare the infected cells with the uninfected control daily for signs of CPE. Change the medium every 5 d, if necessary.
If CPE is evident (usually apparent by ~14 d post-infection), RCA is present in the purified stock. The relative amount of RCA to pfu can be inferred by comparing CPE on the three infected dishes.}

^{xxi. Extract DNA from dishes showing signs of CPE. Analyze by restriction enzyme digestion and agarose gel electrophoresis.
The left end of the RCA genome will have a structure identical to that of wild-type Ad, owing to the presence of E1.
See Troubleshooting.}

^{TROUBLESHOOTING}

^{Problem: There are few or no colonies after cotransformation of BJ5183 cells.}

^{[Step 10]}

^{Solution: Consider the following:}

^{1. The wrong strain of cells may have been used. Ensure that BJ5183 cells are used.}

^{2. The competence of the BJ5183 cells may be too low. Check the competence of the cells and generate new competent cells if necessary, or obtain competent cells from a commercial source.}

^{3. The incorrect antibiotic may have been used, or too much kanamycin may have been used. Ensure that kanamycin was used, and that the concentration is correct (25 µg/mL).}

^{4. The cotransformation conditions may be suboptimal. Try using AdEasier-1 cells, or a more efficient transformation method, such as electroporation.}

^{Problem: There are too many colonies after cotransformation of BJ5183 cells.}

^{[Step 10]}

^{Solution: This is likely caused by incomplete digestion of the shuttle plasmid by PmeI. Consider the following:}

^{1. Use less DNA or more PmeI.}

^{2. Ensure that the PmeI is active.}

^{3. Check the digestion efficiency by agarose gel electrophoresis.}

^{Problem: There is failure to generate plaques on 293 cells after initial transfection.}

^{[Step 26]}

^{Solution: There are a number of possible causes for this. Consider the following:}

^{1. The DNA preparation may not be appropriate. Prepare DNA by CsCl gradient centrifugation and verify DNA concentration before use.}

^{2. There may be incomplete digestion with PacI. Ensure that PacI is active. Examine the PacI digestion efficiency by agarose gel electrophoresis.}

^{3. There may be a defect in the Ad vector backbone. Analyze the plasmid structure of the Ad vector backbone by digestion with several restriction enzymes. If a defect is detected, generate a new clone.}

^{4. The size of the insert may exceed the upper limit of Ad packaging. It may be necessary to choose a different shuttle vector capable of accommodating the insert.}

^{5. The passages of the 293 cells may be too high. Thaw a new aliquot of 293 cells.}

^{6. The transfection efficiency may be too low. Optimize the transfection protocol by trying different amounts of DNA and transfection reagent, or try another transfection reagent or method (e.g., electroporation).}

^{7. The transgene product may be cytotoxic. Use a weaker or inducible promoter.}

^{Problem: No virus band is visible on CsCl gradients.}

^{[Step 40]}

^{Solution: Consider the following:}

^{1. The density of the CsCl solutions is incorrect. Verify the densities by weighing 1 mL of each solution.}

^{2. The two CsCl solutions may have mixed in the step gradient. Overlay the 1.35 g/mL CsCl with the 1.25 g/mL CsCl very carefully. Ensure that a continuous, slow stream of 1.25 g/mL CsCl is ejected and that the phases do not mix.}

^{Problem: No transgene expression is detected.}

^{[after Step 48]}

^{Solution: Consider the following:}

^{1. The transgene or the promoter may be mutated. Analyze purified capsid DNA by restriction analysis and sequencing. If an error is detected, screen other plaque isolates for transgene expression.}

^{2. The transgene may not be expressed efficiently. Verify that the promoter is active in the cell type being used. Ensure that Kozak and polyadenylation sequences have been included in the construct}

^{3. The purified virus could be replication competent. Examine replication on non-complementing cells, such as A549 (see Steps 48.xiii-48.xxi). If virus is replication competent, or if RCA levels are high, purify the vector again, starting from the plaque purification step.}

^DISCUSSION

^{Most Ad vectors are based on the well-characterized human Ad serotypes 2 or 5. The 36-kbp linear, double-stranded DNA genome encodes genes that are classified based on the timing of their expression (Fig. 2 ). Early genes (E1, E2, E3, and E4) are expressed before the onset of DNA replication. Proteins encoded by the early genes function to activate other Ad genes, replicate the viral DNA, interfere with immune recognition of infected cells, and modify the host-cell environment to make it more conducive to viral replication. The late genes (the major late transcription unit, pIX, and IVa2) are expressed after DNA replication and primarily encode proteins involved in capsid production and packaging of the Ad genome. The viral DNA also contains the origins of replication (the inverted terminal repeats [ITR], ~100 bp located at both the left and right ends) and the packaging sequence (~150 bp located immediately adjacent to the left ITR).}

View larger version (8K): [in this window] [in a new window]

Figure 2. Simplified transcription map of the Ad5 genome. See Discussion for details.

^{The commercially available AdEasy system is widely used for recombination-mediated construction of Ad vectors (Fig. 1 ). This consists of several variations of two plasmids (He et al. 1998 ; He 2001 ). The shuttle plasmid contains a kanamycin resistance cassette flanked by the left and right ends of an E1-deleted Ad vector genome, a multiple cloning site located in the E1 locus, and several kilobase pairs of Ad5 DNA downstream from the E1 region (called the right arm of homology). The backbone plasmid encodes the majority of the Ad5 genome and an ampicillin resistance cassette. In vivo recombination between homologous sequences contained in both plasmids transfers the kanamycin resistance cassette, left ITR and packaging sequence, and the transgene from the shuttle into the backbone plasmid, generating an infectious recombinant Ad vector. The choices of shuttle and vector depend on the needs of the investigator, with the primary considerations being the promoter to be used, the size of the insert, and whether vector tracking is desired. Two variations of pAdEasy (the Ad genomic plasmid) are available: pAdEasy-1 has an intact E4 region, whereas in pAdEasy-2, E4 is deleted. Deletion of E4 increases the cloning capacity of resultant vectors, but this requires the use of 911- or 293-based cell lines that express E4.}

^{ACKNOWLEDGMENTS}

^{We thank Robert Lanthier for a critical reading of the manuscript. Research in the Parks laboratory is supported by grants from the Canadian Institutes of Health Research (CIHR) and the Jesse Davidson Foundation for Gene and Cell Therapy, a CIHR/Muscular Dystrophy Canada/Amyotrophic Lateral Sclerosis Society of Canada Partnership Grant, and the Premier’s Research Excellence Award. R.J.P. is a CIHR New Investigator. P.J.R. is supported by a Canada Graduate Scholarship from the National Science and Engineering Research Council.}

^REFERENCES