AN EFFICIENT PROCEDURE TO SELECT AND RECOVER RECOMBINANT ADENOVIRUS VECTORS

Adenoviruses are efficient gene transfer vectors for a variety of cell types. To date, the most widely used methods to construct recombinant adenoviruses involve either in vitro ligation or recombination betwee n one-half of the virus genome, previously cloned in a plasmid vector and engineered to contain the desired expression cassette, and the oth er half of the virus genome prepared from virions. Although quite effe ctive, these approaches produce viral progeny containing a mixture of recombinant and parental background virus. Thus the recovery of the re combinant virus can be difficult especially when it grows more slowly than the parental virus. To improve selection and recovery of recombin ant adenoviruses, we have constructed an adenovirus vector, AdTG6553, in which the E1 region has been replaced by the thymidine kinase (tk) gene of herpes simplex virus type 1. We show that infection of cells w ith AdTG6553 in the presence of the nucleoside analog ganciclovir (GCV ) prevents viral replication. The conditional lethal phenotype introdu ced in AdTG6553 makes it a valuable tool to counter-select parental ba ckground virus in the presence of GCV and isolate replication-deficien t recombinant adenoviruses in which the tk expression cassette has bee n replaced by a new gene.