Characterization of Chinese hamster ovary cells stably transformed by a plasmid with an inducible APRT gene.
A plasmid was constructed by fusion of a selectable mammalian gene, hamster adenine phosphoribosyltransferase (APRT), to the Zn(2+)-inducible sheep metallothionein I (MT I) promoter. This plasmid was used to produce stable Chinese hamster ovary (CHO) cell transformants by electroporation to study the effects of induced gene expression on DNA-mediated transformation. The sheep MT Ia promoter was chosen for these experiments because it regulates gene expression differently than murine MT promoters, exhibiting low basal levels of gene expression in uninduced conditions. We have shown that in the absence of Zn2+, there is very low expression of a sheep MT I-APRT fusion gene in stable CHO cells transformants; induction of APRT mRNA and enzyme activity by Zn2+ produced a "threshold" response, from low basal levels to high induced levels, in Zn2+ responsive stable transformant clones. In electroporation experiments, transformation frequencies were unaffected by Zn2+ treatments during a preselection period, but the presence of Zn2+ during selection increased the recovery of stable transformant clones 8- to 10-fold. All stable transformants analyzed displayed Zn(2+)-inducible APRT enzyme activity. Our results indicate that stable mammalian cell transformants with inducible genes under regulation of the sheep MT I promoter should be useful, because of low basal and high induced expression, for studies in which modulation of transcriptional activity is required.
University of Texas M.D. Anderson Cancer Center, Science Park-Research Division, Smithville 78957.
C A
CA
Walter
R M
RM
Humphrey
G M
GM
Adair
R S
RS
Nairn
D000228:Adenine Phosphoribosyltransferase / Q000235:genetics* / Q000378:metabolism; D000818:Animals; D016466:CHO Cells; D002455:Cell Division; D002461:Cell Line, Transformed; D002872:Chromosome Deletion; D002999:Clone Cells; D006224:Cricetinae; D008668:Metallothionein / Q000235:genetics; D010957:Plasmids; D011401:Promoter Regions, Genetic; D015183:Restriction Mapping; D014162:Transfection
D008668:Metallothionein; D000228:Adenine Phosphoribosyltransferase