Hepatocyte-specific deletion of the keap1 gene activates Nrf2 and confers potent resistance against acute drug toxicity
Section snippets
Materials and methods
Generation of mice with floxed keap1-targeted allele and hepatocyte-specific disruption of the keap1 gene. Mouse keap1 genomic DNA was isolated from mouse 129SvJ genomic library (Stratagene). To construct a targeting vector, we included cDNAs encoding Diphtheria toxin A (DTA) and neomycin resistance (Neo) for negative and positive selection, respectively. We constructed a triple-loxP keap1-targeting vector to generate a floxed mouse keap1-targeted locus consisting of a loxP site inserted into
Generation of hepatocyte-specific keap1-null mutant mice
To examine how constitutive activation of Nrf2 modulates the defense mechanisms against xenobiotic toxicity, we generated hepatocyte-specific keap1-null mutant mice using the Cre-loxP system (Fig. 1). A targeting vector was constructed in which exons 4–6 of the keap1 locus were flanked with loxP sequences (Fig. 1A). This region encodes the double glycine repeat (DGR) domain, a region that is important for Keap1 interaction with Nrf2 [29], [30]. It was expected therefore that Cre-loxP
Discussion
In this study, we observed that somatic disruption of keap1 does not interfere with the development or the morphological and physiological integrity of the liver. Our microarray analysis showed that genetic disruption of the keap1 gene induced overlapping but clearly distinct sets of drug-metabolizing enzymes from those induced by exogenous chemicals. Through the induction of detoxifying enzymes, specific knockout of the keap1 gene confers hepatocytes with a strong resistance to drug-induced
Acknowledgments
We thank Drs. K. Araki and K. Yamamura for providing us AYU1-Cre transgenic mice and Dr. S. Yamamoto for giving us advice on microarray data interpretation. We also thank Ms. N. Kaneko and R. Kawai for experimental assistance and Dr. V. Kelley for English edition. This work was supported by grants from the Ministry of Education, Science, Sports and Culture (H.M. and M.Y.), the Uehara Memorial Foundation (H.M.), Yamanouchi Foundation (H.M.), Atherosclerosis Foundation (M.Y.), and ERATO-JST
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