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Genomic clones corresponding to Cyp1b1 were obtained by screening a 129/Sv genomic library (CLONTECH) by using the rat Cyp1b1 cDNA as a probe. A clone spanning 10.5 kb and containing two of the three exons of the murine Cyp1b1 gene (exon 1 is noncoding) was subcloned as a SalI fragment into pGEM-3Z (Promega). Both coding exons were targeted to disrupt the gene. The targeting vector was created by inserting a 1.7-kb cassette containing the bacterial phosphoribosyltransferase II gene under control of the phosphoglycerate kinase-1 promoter (PGK-NEO), which confers resistance to the aminoglycoside G418 (Life Sciences, St. Petersburg, FL), into the HincII site of exon 3. This cassette was excised from pPNT (PMID: 2065352) and was inserted in the opposite transcriptional orientation as the Cyp1b1 gene. As a negative selection against random integration of the construct, the herpes simplex virus thymidine kinase gene was inserted at the 5' end of the targeting vector (Bradley, A., 1987 Teratocarcinoma and Embryonic Stem Cells: A Practical Approach - IRL, Oxford) . The construct containing 4.4 kb of 5'- and 1.5 kb of 3'-genomic DNA flanking the PGK-NEO cassette was made in four cloning steps: (i) The genomic clone, designated 24.7, in the SalI site of the polylinker region of pGEM-3Z was digested with BamHI, which cleaves once in the pGEM-3Z polylinker region and once in the genomic clone, and was gel-purified and religated. (ii) The shortened genomic clone then was digested with HindIII, which also cleaves once in the pGEM-3Z polylinker region and once in the genomic clone, and was gel-purified and religated. Doing so also removed the HincII site in the polylinker region of pGEM-3Z. (iii) The 5'- and 3'- shortened genomic clone (24.7BH) was digested with HincII and was ligated with a PGK-NEO cassette that was isolated from pPNT with XhoI-XbaI and made blunt-ended with Klenow polymerase. This ligation recreated a XbaI site 5' of the PGK-NEO cassette that was used in a diagnostic Southern blot. (iv) The targeted gene (24.7BH/NEO) was excised with BamHI and HindIII from pGEM-3Z and was gel-purified and ligated to pMC1TK (PMID:3194019), which was digested with BamHI and HindIII. The second exon was targeted by a vector in which a 600-bp XhoI fragment spanning part of exon 2 and intron 2 was deleted and replaced by pMC1NeopolyA (Stratagene) in the opposite transcriptional orientation as the Cyp1b1 gene. The herpes simplex virus thymidine kinase gene also was added 5' of this construct as described above.
Homozygous: To determine whether the results in vitro were predictive for sensitivities to DMBA in vivo, carcinogen bioassays were performed. DMBA was administered by gavage at a dosage level and frequency that induces tumors in normal mice (PMID:9067556). At 6 months after treatment, 60% of the wild-type mice had died from tumor burdens or had been killed because of severe morbidity whereas <10% of the Cyp1b1-null mice had died. Autopsies revealed that 70% of wild-type mice developed lymphomas whereas only 7.5% of the Cyp1b1-null mice had these tumors throughout the course of the study. A quarter of the mice also exhibited skin abnormalities with hyperplasia and various tumors in the most extreme cases. These abnormalities rarely were seen in the Cyp1b1-null mice. Occasionally, tumors in ovaries were seen in wild-type mice. The Cyp1b1-null mice had a higher level of lung adenocarcinomas as compared with wild-type mice (12.5 vs. 2.4%). However, it should be noted that these tumors were found at the end of the study, at which time only 40% of the wild-type mice were left for autopsy. Thus, if adenocarcinomas arise, they may be underestimated in the wild-type mice because of early mortality caused by the lymphomas. These data indicate that, for this oral route of administration, Cyp1b1 is required for the activation of DMBA to carcinogenic metabolites that produce lymphomas.Heterozygous: Not characterized.
The plasmid DNA used for targeting was purified by banding twice on cesium chloride. After linearization by ClaI, 5, 10, 20 or 40 ug of DNA were electroporated into Genome System (St. Louis) embryonic stem (ES) cells by using conditions previously described (PMID:7539101). The cells were grown on gamma-irradiated G418-resistant mouse EFs in the presence of 1,000 units of lymphocyte inhibitory factor/ml (ESgro, Life Sciences). ES cell clones resistant to G418 and ganciclovir (a gift from Syntex, Palo Alto, CA) were selected and screened for homologous recombination. Clones having the expected Southern blot pattern for homologous recombination (see below) were regrown and injected into C57BL/6N blastocysts. The blastocysts were transferred into the uterus of a pseudopregnant recipient NIH Swiss mouse to produce animals showing chimerism. Male chimeras presenting >75% 129/Sv contribution, as determined by coat color, were bred with C57BL/6N females to determine whether the disrupted allele was transmitted to the germline. Homozygotes were produced by crossing the F1 generation. These mice were then crossed with the 129S6/SvEvTac to create a mixed background mouse strain. The mice were then backcrossed to 129S6/SvEvTac for 6 generations and made homozygous null at that point by brother:sister mating
A sample from this MMRRC strain was analyzed using the Mouse Universal Genotyping Array (MUGA) and MMRRC computational tools were used to assess the genetic background. A summary of the data can be found here.
Wild-type littermates
Buters JT, Sakai S, Richter T, Pineau T, Alexander DL, Savas U, Doehmer J,Ward JM, Jefcoate CR, Gonzalez FJ. Cytochrome P450 CYP1B1 determinessusceptibility to 7, 12-dimethylbenz[a]anthracene-induced lymphomas. Proc NatlAcad Sci U S A. 1999 Mar 2;96(5):1977-82. (Medline PMID: 10051580)
Colony Surveillance Program and Current Health Reports
Limited quantities of breeder mice (recovered litter) are available from a cryoarchive; recovered litter usually available to ship in 3 to 4 months.
Cryopreserved material may be available upon request, please inquire to mmrrc@med.unc.edu for more information.
Distribution of this strain requires submission of the MMRRC Conditions of Use (COU). A link to the COU web form will be provided via email after an order has been placed; the form should be completed then or the email forwarded to your institutional official for completion.
The donor or their institution limits the distribution to non-profit institutions only.
Additional charges may apply for any special requests. Shipping costs are in addition to the basic distribution/resuscitation fees. Information on shipping costs and any additional charges will be provided by the supplying MMRRC facility.
1 The distribution fee covers the expense of rederiving mice from a live mouse; you will receive the resulting litter. The litter will contain at minimum one mutant carrier; the actual number of animals and the gender and genotype ratios will vary. (Typically, multiple breeder pairs can be established from the recovered litter.) Prior to shipment, the MMRRC will provide information about the animals recovered. If you anticipate or find that you need to request specific genotypes, genders or quantities of mice in excess of what is likely from a resuscitated litter, you may discuss available options and pricing with the supplying MMRRC facility.
2 An aliquot contains a sufficient number of embryos (in one or more vials or straws and based on the transfer success rate of the MMRRC facility) to transfer into one to three recipients. The MMRRC makes no guarantee concerning embryo transfer success experienced in the recipient investigator's laboratory. Neither gender nor genotype ratios are guaranteed.
3 An aliquot is one straw or vial with sufficient sperm to recover at least one litter of mice, as per provided protocols, when performed at the MMRRC facility. The MMRRC makes no guarantee concerning the success of these procedures when performed outside the MMRRC facilities.
4 The distribution fee covers the expense of resuscitating mice from the cryo-archive; you will receive the resulting litter. The litter will contain at minimum one mutant carrier; the actual number of animals and the gender and genotype ratios will vary. (Typically, multiple breeder pairs can be established from the recovered litter.) Prior to shipment, the MMRRC will provide information about the animals recovered. If you anticipate or find that you need to request specific genotypes, genders or quantities of mice in excess of what is likely from a resuscitated litter, you may discuss available options and pricing with the supplying MMRRC facility.