Stable expression of rat cytochrome P450IA2 cDNA and hydroxylation of 17β-estradiol and 2-aminofluorene in V79 Chinese hamster cells
✍ Scribed by Catherine Wölfel; Karl-Ludwig Platt; Satish Dogra; Hansruedi Glatt; Felix Wächter; Johannes Doehmer
- Publisher
- John Wiley and Sons
- Year
- 1991
- Tongue
- English
- Weight
- 820 KB
- Volume
- 4
- Category
- Article
- ISSN
- 0899-1987
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✦ Synopsis
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 P450IA2. Full-length cDNA encoding rat P450IA2 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 P450IA2-specific enzymatic activities such as hydroxylation of 17 beta-estradiol and 2-aminofluorene.