Abstract
To investigate molecular events regulating the transcription of genes inducible by phenobarbital, transgenic mouse strains were developed incorporating rat cytochrome P450 2B2 (CYP2B2) genes. Expression in mouse tissues was analyzed for two series of rat CYP2B2 gene constructs, of 19 and 39 kilobase pairs total length, each containing the entire coding region, introns, and 3'-flanking sequences of CYP2B2, but differing in the respective lengths of 5'-flanking sequence. One group of mice, whose transgene included the complete 2B2 gene but only 800 base pairs of 5'-proximal sequence, were not phenobarbital-inducible in mouse liver or in any extrahepatic tissue; rather, these genes were expressed at very high levels constitutively and selectively in only kidney and liver. A second group of mice with an identical transgene, except for the presence of an additional 19 kilobase pairs of 5'-flanking sequence, expressed 2B2 only in the liver and at high levels only after phenobarbital treatment, analogous to the expression pattern observed for the endogenous CYP2B2 gene in the rat. These results demonstrate that, in vivo, phenobarbital induction and tissue-specific control requires interaction of regulatory elements far upstream of the core CYP2B2 promoter region and upstream of motifs indicated previously as determinants of phenobarbital responsiveness.
Highlights
To investigate molecular events regulating the tran- certainglutathione S-epoxide transferases, UDP-glucuronyl scription of genes inducible by phenobarbital,trans- transferases,aldehyde dehydrogenases, and the cytochrome genic mouse strains were developed incorporating rat P450 monooxygenases [1]
Alphenobarbital-induciblein mouse liver or in any extra- though it wasdemonstrated previously that PBstimulates hepatic tissue; rather, these genes were expressed at these genes at the transcriptional level
Two mouse colonies were founded which contained the rat CYP2B2 transgenes with 20 kb of 5“genetic sequence; estimates by Southern blot phosphorimaging indicatedthat colony 39E-B possessed a single copy of the transgene while colony 39E-Y maintained -3 copies (Table I)
Summary
Hydrocarbons, peroxisome proliferator compounds, and several Gene Fragments-CYP2B2 clone 47B was isolated previously froma sedative-hypnoticmedications. Isolated from a Sprague-Dawley rat genomic library cloned into the cosmid vector, pWE15 (Clontech, Palo AClAto), using a random-primed ~x-~~P-labeXlbeadI-AccIfragment derived from clon4e2 spanning -346 bp to +10 bp, relative to the transcription start site (Fig.).All clones were identified and characterizebdy restriction digestion, hybridization to specific oligomers, and by direct sequence analysis. Gene Constructs-Clone 47B was digested with BamHI, electrophoresed ina low-melting point agarose (LMA) gel, athnde 16-kilobasepair (kb)fragment, encompassing exon 4 to 4 kb 3’ of exon 9 plus 4-kb of phage DNA, was excised and purified with GELase (Epicenter, Madison, WI), phenol-extracted, and ethanol-precipited. The plasmid, pBS‘-19E, was identical across the CYP2B2 gene sequence as pWE‘-39E, except that the former contained only 0.8 kb of 5”flanking sequence.The pBS”19E insert was used to generate the 19E mouse lines.
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