Characterization of Chinese hamster ovary cells stably transformed by a plasmid with an inducible APRT gene

Christi A. Walter, Ronald M. Humphrey, Gerald M. Adair, Rodney S. Nairn

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Abstract

A plasmid was constructed by fusion of a selectable mammalian gene, hamster adenine phosphoribosyltransferase (APRT), to the Zn2+-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 Zn2+-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.

Original languageEnglish (US)
Pages (from-to)208-216
Number of pages9
JournalPlasmid
Volume25
Issue number3
DOIs
StatePublished - May 1991
Externally publishedYes

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ASJC Scopus subject areas

  • Molecular Biology

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