( ne from the pAdEasy-1 vector for assemb...)Generate Adenovirus Vectors in E. coli by Homologous Recombination,,,with the AdEasy Adenoviral Vector System

ne from the pAdEasy-1 vector for assembly of replication-defective adenovirus. To confirm the presence of infectious virus, primary viral stocks were subsequently used to transduce HEK293 cells. Three days after transduction, cells were analyzed for expression of their respective reporter genes. Cells transduced with AdCMV-GFP and Ad-CG (both harboring a GFP reporter gene) were visualized by fluorescence microscopy. Cells transduced with AdCMV-LacZ were stained with X-gal using Stratagenes In Situ b-Galactosidase Staining Kit. From representative photographs of HEK293 cells transduced with the test vectors (Figure 4), the positive identification of the reporter gene expression in transduced cells indicates that infectious adenovirus were produced using the AdEasy vector system.

Fig.4

Conclusions

Stratagenes AdEasy vector system simplifies and speeds up the process of generating a recombinant adenovirus, compared to traditional methods of preparation. With the AdEasy systems unique two-step process, a gene of interest, or expression cassette, is subcloned into a shuttle vector, then transferred into the adenoviral genome by homologous recombination in E. coli. This method eliminates the need for laborious plaque isolations, since the resulting adenoviral plasmid can be directly transfected into HEK293 cells to prepare adenovirus. With the AdEasy system, adenoviral-mediated gene transfer and expression are realized efficiently and conveniently.

REFERENCES

1. Benihoud, K., Yeh, P., and Perricaudet, M. (1999) Curr. Opinion in Biotech. 10: 440-447.

2. Berkner, K. (1988) Biotechniques 6: 616-625.

3. He, T., et al. (1998) Proc. Natl Acad Sci USA 95: 2509-251
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