Transduction of the Chinese hamster ovary aprt gene by herpes simplex virus

Abstract

The Chinese hamster ovary adenine phosphoribosyl transferase gene (aprt) was reengineered to be flanked by sequences from the thymidine kinase (tk) gene of herpes simplex virus. This construct was cotransfected with DNA from herpes simplex virus type 1, and after 3 days, virus was harvested and Tk- plaques were selected after the virus was plated on Tk- cells in the presence of bromodeoxycytosine. Recombinant viruses were identified by dot-blot hybridization, and the arrangement of aprt and tk sequences were determined by Southern blot hybridization. Analysis of the recombinants revealed that acquisition of aprt sequences resulted from insertional inactivation of the tk locus as a consequence of homology-based recombination. Recombination was precise, as evidenced by the failure to detect plasmid sequences or the synthetic restriction endonuclease sites that bounded the mutant tk gene in the aprt-tk construct. Infection of Aprt- mouse or Chinese hamster ovary cells with UV-irradiated virus and selection in medium containing azaserine and adenine resulted in the survival of numerous colonies that stably express the aprt gene. Transformed cells synthesized an aprt mRNA that is identical to wild-type mRNA as determined by Northern blot and S1 nuclease analyses. Cells lytically infected with the recombinant virus do not appear to transcribe the aprt gene. Thus, infected cells differentiate between virus and foreign promoters even when a cellular gene is cis to the virus chromosome.