AdEasy Just Got Easier

T.-C. He, Kenneth W. Kinzler, and Bert Vogelstein

Use of AdEasier-1 Cells (also termed BJAdEasy Cells or AdEasier Cells) for Generating Adenoviral Recombinants

We are now sending AdEasier-1 cells to investigators who request other components of the system (or to anyone who asks us for them).  AdEasier-1 cells are BJ5183 derivatives which already contain the AdEasy-1 plasmid.  In order to generate recombinant vectors ready to transfer into 293 cells, one simply has to transform the AdEasier-1 cells with a shuttle vector (such as pAdTrack-CMV) containing the gene of interest, and then proceed as with the standard AdEasy system.  

Because BJ5183 cells have a relatively high frequency of homologous recombination, unwanted or detrimental rearrangements and/or recombinations of the pAdEasy-1 sequences in AdEasier-1 cells can occur. It is thus  important to pick individual clones and characterize the clones with extensive restriction digestions, usually with Hind III and/or Pst I.   The digestion pattern can be compared with the pAdEasy-1 stock DNA made in a non-recombinant strain (like DH10B). A restriction digest characterization should optimally be carried out on DNA from the large-scale culture that is used to prepare competent cells.  Stratagene is also selling these cells (which they call BJ5183-AD-1) in competent form, eliminating the need for picking clones, checking them, and making them competent.  For investigators planning to make only a few adenoviral constructs, this is particularly convenient.  

Advantages of Using AdEasier-1 Cells:

1) The cells are extremely efficient for generating recombinants.  Up to 100 x more recombinants are formed compared to the original AdEasy system employing cotransfection of BJ5183 cells.  

2) It does not require preparation of high quality pAdEasy plasmids.

3) It is probably possible to generate recombinants by using conventional chemical transformation methods       instead of electroporation, though we haven't explored this.

Note that Zeng et al. have independently described the generation and utility of such cells (Zeng M, Smith SK, Siegel F, Shi Z, Van Kampen KR, Elmets CA, Tang DC.  AdEasy system made easier by selecting the viral backbone plasmid preceding homologous recombination. Biotechniques 31: 260-262 (2001)).

For investigators who would like to make their own versions or derivatives of AdEasier-1 cells from BJ5183 cells, the following method can be used:

     1)      Transform 50 ng of pAdEasy-1 plasmid into BJ5183 cells and plate the transformation mix on 

            agar plates conferring resistance to both ampicillin and streptomycin.

2)      Pick 10-20 colonies and grow each in 2 ml LB/Amp/Strep medium with continuous shaking at 37⁰C, overnight.

3)      Purify the DNA from each of the cultures (see Appendix E: Alkaline Lysis Protocol for Plasmid Minipreps).

4)      Use 20-30% of the miniprep DNA for restriction digestion (Hind III, Pst I, etc) to confirm integrity of clones.  Pick one confirmed clone for subsequent use.

5)      Grow the confirmed clone in LB/Amp/Strep medium.

6)      Prepare electrocompetent cells using the protocol described in the Appendix

7)      Transform the Pme I-linearized shuttle plasmid into the electrocompetent cells;

8)      Follow the rest of the protocol described in the Practical Guide;