Stable expression of rat cytochrome P450IA2 cDNA and hydroxylation of 17 beta-estradiol and 2-aminofluorene in V79 Chinese hamster cells.
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.
Wölfel
C
C
Institut für Toxikologie, Johannes Gutenberg-Universität, Mainz, Germany.
Platt
K L
KL
Dogra
S
S
Glatt
H
H
Wächter
F
F
Doehmer
J
J
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015152:Blotting, Northern; D015139:Blotting, Southern; D002460:Cell Line; D003001:Cloning, Molecular; D006224:Cricetinae; D003412:Cricetulus; D019388:Cytochrome P-450 CYP1A2; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics* / Q000378:metabolism; D004247:DNA / Q000235:genetics; D004958:Estradiol / Q000378:metabolism; D005449:Fluorenes / Q000378:metabolism; D015870:Gene Expression; D005822:Genetic Vectors; D006900:Hydroxylation; D066298:In Vitro Techniques; D008969:Molecular Sequence Data; D010088:Oxidoreductases / Q000235:genetics* / Q000378:metabolism; D051381:Rats; D011994:Recombinant Proteins / Q000378:metabolism; D014162:Transfection
D005449:Fluorenes; D011994:Recombinant Proteins; C012177:2-aminofluorene; D004958:Estradiol; D004247:DNA; D003577:Cytochrome P-450 Enzyme System; D010088:Oxidoreductases; D019388:Cytochrome P-450 CYP1A2