Biochemical and Biophysical Research Communications
Enhancer elements in the mouse Cypla2 gene for constitutive expression☆
Section snippets
Materials and methods
Chemicals. Materials for culturing hepatocytes were purchased from Wako Pure Chemical Industries (Osaka, Japan), Gibco BRL (Grand Island, NY), and Sigma Chemical (St. Louis, MO). Percoll was obtained from Amersham Pharmacia Biotech (Uppsala, Sweden). TaKaRa LA-Taq and native Pfu DNA polymerase were from TaKaRa Biochemicals (Shiga, Japan) and Stratagene (La Jolla, CA), respectively. BigDye terminator cycle sequencing ready reaction kit was from Perkin–Elmer Applied Biosystems (Foster city, CA)
Results and discussion
To investigate the transcriptional activity of the isolated mouse Cyp1a2 gene, we constructed a reporter gene (−4795/+27Luc) fusing 4.8 kbp of the 5′-flanking region of mouse Cypla2 with the 5′-end of the luciferase structure gene. This reporter gene was transiently transfected into mouse hepatocytes by electroporation, showing that −4795/+27Luc had prominent transcriptional activity. To further identify the regulatory region in the 5′-flanking 4.8 kbp sequence, various 5′-deletion mutants were
Acknowledgements
This work was partly supported by Grants-in-Aid from the Japanese Ministry of Education, Culture, Sports, Science and Technology, and from the Smoking Research Foundation.
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Abbreviations: AhR, aryl hydrocarbon receptor; Arnt, AhR nuclear translocator; AP-1, activator protein 1; (k)bp, (kilo)base pairs; EMSA, electrophoretic mobility shift assay; TCDD, 2,3,7,8-tetrachlorodibenzo-p-dioxin; TPA, phorbol-12-O-tetradecanoate-13-acetate; TRE, TPA-responsive element; cTRE, consensus TRE; XRE, xenobiotic-responsive element.