Transduction of the CHO aprt 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 CHO aprt structural gene linked to a Moloney murine sarcoma virus (MSV) promoter into the E3 region of adenovirus-5. The CHO aprt gene was in the opposite orientation to the adenovirus E3 promoter. Mouse Lapt- tk- (LAT) cells expressed the CHO aprt 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 approximately 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 CHO aprt-specific DNA and with viral DNA probes. The results indicated that the CHO aprt gene was integrated into the LAT cells at a site other than mouse aprt. Although neighboring viral sequences were integrated and maintained in the transductants, viral sequences further upstream and downstream of the aprt gene were absent.
Konan
V
V
Department of Biology, Indiana University, Bloomington.
Sahota
A
A
Graham
F L
FL
Taylor
M W
MW
D000225:Adenine / Q000378:metabolism; D000228:Adenine Phosphoribosyltransferase / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism; D000260:Adenoviruses, Human / Q000201:enzymology / Q000235:genetics*; D000818:Animals; D015139:Blotting, Southern; D006224:Cricetinae; D003412:Cricetulus; D005260:Female; D006367:HeLa Cells; D006801:Humans; D007739:L Cells / Q000378:metabolism* / Q000382:microbiology; D051379:Mice; D010053:Ovary; D011995:Recombination, Genetic; D014161:Transduction, Genetic
D000228:Adenine Phosphoribosyltransferase; D000225:Adenine