Transduction of the CHOaprt gene into mouse L cells using an adeno-5/APRT recombinant virus

An adenovirus-5 recombinant virus Adapt1 carrying the Chinese hamster ovary (CHO) adenine phosphoribosyltransferase (aprt) gene was constructed by insertion of a 2.5-kb fragment containing the complete CHOaprt structural gene linked to a Moloney murine sarcoma virus (MSV) promoter into the E3 region of adenovirus-5. The CHOaprt gene was in the opposite orientation to the adenovirus E3 promoter. Mouse Lapt− tk− (LAT) cells expressed the CHOaprt gene when infected with the virus, even at low MOI (O.1). APRT activity was detectable from approximately 20 h postinfection. At a low frequency, LAT cells were transformed to aprt+, and four stable transductants were selected in adenine, azaserine (AA) medium. Such cells expressed APRT at ∼50% wild-type activity and the enzyme was shown to be CHO APRT by starch gel electrophoresis. DNA was isolated from the transductants and probed with CHOaprt-specific DNA and with viral DNA probes. The results indicated that the CHOaprt gene was integrated into the LAT cells at a site other than mouseaprt. Although neighboring viral sequences were integrated and maintained in the transductants, viral sequences further upstream and downstream of theaprt gene were absent.