HIGH-THROUGHPUT METHOD FOR CREATING AND SCREENING RECOMBINANT ADENOVIRUSES

Replication defective adenoviruses are being considered as vectors in therapeutic applications of gene therapy, as well as research fools in studying gene function. Important to their successful utilization is the development of techniques to isolate new recombinants quickly, whi ch are not contaminated with wild-type virus. We describe a modificati on of the traditional technique to create recombinant adenoviruses in which a 5' plasmid containing vector sequence is cotransfected into 29 3 cells with viral DNA. In our protocol, the viral DNA is derived from the 3' portion of an E1-deleted recombinant that expresses the green fluorescent protein. Visualization of the cotransfection by fluorescen t microscopy distinguishes recombinant plaques (nonfluorescent or 'whi te plaque') from background plaques (green fluorescent or 'green plaqu e'). Using this approach we have been able to increase substantially t he success and throughput for creating new recombinants while minimizi ng contamination. This has been used to isolate adenoviral vectors del eted in a number of essential genes.