II. General Considerations

1. Because PmeI and PacI sites are designed to linearize the final constructs for transformation and transfections, avoid using these sites in you inserts. (If you absolutely cannot avoid Pme1 and Pac1 sites, you can still use these vectors, but with more difficulty (employing partial digestions or digestion with EcoRI and recA-assisted restriction endonuclease (RARE) cleavage). 2. Avoid cloning elements that are present more than once in the vector (e.g., CMV promoters) in head-to-head orientations. 3. Except for pAdEasy-1 and pAdEasy-2, all other constructs (including recombinant adenoviral plasmids) confer resistance to kanamycin (NOT ampicilin). 4. High competence of BJ5183 cells is important to obtain homologous recombinants  because these cells have relative low transformation efficiency.   Therefore it is important to carefully follow the protocol for preparation of these cells. 5. Purification of plasmid DNAs: Standard CsCl gradient purification for all plasmid DNAs used in the experiments (especially pAdEasy-1 and pAdEasy-2) is recommended, though miniprep DNAs made by commercial kits (e.g. Nucleobond) may also be acceptable (see FAQ's section). 6. References:
        T.-C. He, et al (1998) PNAS  95: 2509-2514.
        H. Hermeking, et al (1997)  Molecular Cell  1:3-11.
        T.-C. He, et al (1998) Science 281(5382):1509-12
        T.-C. He, et al (1999)  Cell  99:1-20.

III. Generation of Recombinants in Bacterial Cells
  1. Prepare electrocompetent BJ5183 cells in 20 microliters/tube aliquots (see Appendex A and B). 2. Linearize the shuttle plasmids with Pme I. 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 microliters of dd H₂O. 3. Co-transform Pme I-digested shuttle plasmid with 1.0 microliter of adenoviral backbone vector (100 ng/ul). Twenty microliters of electrocompetent E. coli BJ5183 cells were added and electroporation was performed in 2.0 mm cuvettes at  2,500V, 200 Ohms, and 25 micro-FD in a Bio-Rad Gene Pulser electroporator. Resuspend transformation mix in 500 microliters of L-broth (incubation at 37℃ for 10-20 min is optional, depending on background, see FAQ's). Plate on 3 to 5 LB/Kan plates, and grow at 37℃ overnight (16-20 hrs). 4. Pick up 10 to 20 smallest colonies, and grow them in 2 ml L-broth containing 25 microgram/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. Restriction digest DNA from clones with Pac I.  Candidate clones usually yield a large fragment (near 30 kb), plus a smaller fragment of  3.0kb or 4.5kb. 7.  Re-transform two microliters of the correct recombinant miniprep DNA into DH10B (or your favorite plasmid propagation strain). Plasmids are purified by CsCl-banding (see Appendix D) or by using commercially available purification kits for transfection of 293 cells.  

 NOTE:  A more efficient approach to generating adenoviral recombinants has recently been described in the section “AdEasy Got Easier”.

IV. 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 standard Lipofectamine transfection according to manufacture’s manual.  Mix 4 µg of PacI-digested plasmid and 20 µl of Lipofectamine (GIBCO BRL) for each T-25 in 500 µl of OptiMem I medium, and incubate at room temperature for 15-30 min.
4. While waiting, remove growth medium from recipient cells and wash them once with 4 ml serum-free medium (e.g. plain DMEM, or Hank’s).  Add 2.5 ml OptiMem I per T-25 flask. Return to 37℃ CO₂ incubator for ~10 min.
5.  Add Lipofectamine-DNA mix to the flasks, and return to 37oC CO₂ incubator.
6. Remove medium containing Lipofectamin-DNA mix four hours later, and add 6 ml fresh complete DMEM (10% FBS, 1% Pen/Strep). Do not change the lipo/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℃ 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.

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