A SYSTEM FOR RAPID GENERATION OF RECOMBINANT ADENOVIRUSES BY LIGATION

Background: Recombinant adenoviruses provide a versatile tool for gene expression, and both ease of production and ability to infect non dividing cells have led to their widespread use in arthritis models. Classically, their construction is time-consuming, requires homologous recombination in permissive cells, and, despite recent technical improvements such as homologous recombination in bacteria, their construction remains often difficult with manipulation of cosmids or large plasmids containing full-length adenoviral genome.Objectives: To design a simpler systemfor the construction of adenovirus which would allow us to use recombinant adenoviruses to screen the effects of numerous cytokines and their respective inhibitors in arthritis models using a gene therapy approach.Results: We have designed and constructed a system where recombinant adenoviruses are constructed by a simple ligation. Ad-P-EGFP is a recombinant first generation E1-deleted adenovirus where a unique and rare restriction site (PacI) has been engineered at 3.7mu. The shuttle vector (pC5) also contains a PacI site 3 prime of a CMV-driven expression cassette. pC5 only contains the left ITR and packaging signal from wild-type adenovirus serotype 5, but no regions for homologous recombination. This strategy allows for a much smaller shuttle than usual, making manipulation and subcloning of transgene easier. Once the gene of interest cloned in the expression cassette of pC5, the resulting plasmid is digested with PacI and SwaI (another rare cutter engineered just in front of the left ITR) to free a fragment containing all the necessary elements for the construction of a recombinant adenovirus (left ITR, packaging signal and expression cassette). Another engineered PacI site in the backbone of pC5 allows for easier purification. This fragment is then ligated overnight to unpurified PacI restricted and dephosphorylated Ad-P-EGFP viral DNA. The mixture is then directly lipofected without further manipulation in 293 cells. Cells are covered with an overlay and, after 5 to10 days, plaques are screened under a fluorescent microscope and non fluorescent plaques picked and amplified. Typical ratio for fluorescent to non fluorescent plaques are between 1:4 to 1:20.Conclusion: Our system allows for rapid generation of recombinant adenoviruses using common techniques such as ligation and lipofection. It doesn't need homologous recombination, special strain of bacteria, extraction of large DNA fragment or manipulation of cosmids. This system has allowed us to construct rapidly more than 15 different viruses which will be used in arthritis models.Citation: , volume , supplement , year 2002, page Session: Cytokines and inflammatory mediators