Balancer-Cre transgenic mouse germ cells direct the incomplete resolution of a tri-loxP-targeted Cyp1a1 allele, producing a conditional knockout allele

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Abstract

To generate conditional alleles, genes are commonly engineered to contain recognition sites for bacteriophage recombinases, such as Cre recombinase. When such motifs (lox sites) flank essential gene sequences, and provided that Cre recombinase is expressed, Cre recombinase will excise the flanked sequence—creating a conditional knockout allele. Targeted conditional alleles contain a minimum of three lox sites. It would be desirable to have Cre recombinase perform partial resolution (i.e., recombination some of the time between only the two lox sites flanking the marker gene). Here we report use of the commercially available Balancer2-Cre transgenic mouse line to carry out this function from a tri-loxP-site-containing cytochrome P450 1A1 (Cyp1a1) targeted allele. Such incomplete resolution of this complex locus occurred progressively with age in germ cells of male mice; the conditional Cyp1a1 gene was recovered in offspring from mice containing the targeted Cyp1a1 allele and the Cre recombinase transgene. Removal of the marker gene resulted in a conditional Cyp1a1 allele whose expression was indistinguishable from that of the wild-type allele.

Section snippets

Materials and methods

Mice. Balancer2-Cre transgenic mice, having the strain name TgN(balancer2)3Cgn, were purchased from The Jackson Laboratory (Bar Harbor, ME). Generation of the conditional Cyp1a1 knockout mouse strain has been previously described [13]. All experiments involving mice were conducted in accordance with the National Institutes of Health (NIH) standards for the care and use of experimental animals and the University of Cincinnati Institutional Animal Care and Use Committee.

DNA preparations. DNA for

Importance of CYP1A1 in toxicity and carcinogenesis

CYP1A1 is one of the three members of the mammalian CYP1 family, which includes CYP1A1, CYP1A2, and CYP1B1 [25]. The CYP1A1 gene is highly inducible at the level of transcription, the up-regulation being controlled by the aromatic hydrocarbon receptor (AHR). Translocation of the cytoplasmic AHR, to become a nuclear transcriptional activator, occurs as the result of ligand binding. Ligands for the AHR include numerous planar foreign chemicals and, in general, AHR ligands are metabolized by

Conclusions

The Nes/Cre transgene present in the bal2Cre line of transgenic mice has been used by others to bypass embryonic lethality associated with essential conditional floxed alleles [22]. This result reflects a mosaic pattern of Cre-mediated recombination in tissues—some cells showing recombination, others not. This mosaic pattern is the result of threshold expression of Cre recombinase such that incomplete resolution of loci containing three or more lox sites may be observed and inherited. One

Acknowledgements

We thank our colleagues for many fruitful discussions and a careful reading of the manuscript. This work was supported, in part, by NIH Grants R01 ES08147 and P30 ES06096.

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    Present address: Department of Biochemistry, Nihon University School of Medicine, 30-1 Oyaguchikami-cho, Itabashi-ku, Tokyo 173-8610, Japan.

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