Stable expression of rat cytochrome P450IA2 cDNA and hydroxylation of 17β‐estradiol and 2‐aminofluorene in V79 Chinese hamster cells

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Abstract
In continuation of our work toward the establishment of a working cell bank for metabolic and toxicological studies, V79 Chinese hamster cells were genetically engineered for stable expression of rat cytochrome P4501A2. Full‐length cDNA encoding rat P4501A2 was obtained by searching a cDNA library made from Aroclor 1254‐induced rat liver mRNA and by joining a small 5′‐end fragment to a fragment containing the rest of the cDNA. The sequence of the cDNA was confirmed by DNA sequencing and comparison to a previously published cDNA sequence. The reconstructed full‐length cDNA was inserted into a simian virus 40 early promoter‐containing eukaryotic expression vector and cotransferred with the neomycin phosphotransferase gene as a selective marker into V79 cells by the calcium/phosphate‐coprecipitation technique. G418‐resistant V79 cell clones were checked for chromosomal integration of the cDNA by Southern blotting, for expression of authentic mRNA and protein by northern and western blotting, and for P4501A2‐specific enzymatic activities such as hydroxylation of 17β‐estradiol and 2‐aminofluorene.