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 (16) 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 (17). 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 (18), 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 (Jeroen T. M. Buters, Shuichi Sakai, Thomas Richter, Thierry Pineau, David L. Alexander, Uzen Savas, Johannes Doehmer, Jerrold M. Ward, Colin R. Jefcoate, and Frank J. Gonzalez. PNAS, Vol. 96, Issue 5, 1977-1982, March 2, 1999).

RW-4 derived from 129X1/SvJ

Homozygous: The pathology of aging CYP1B1 null mice on a B6; 129 background was studied in groups of 29 males and 30 females. By 12 months, 50% of the female mice had developed a unusual progressive glomerulonephritis while males had similar renal lesions later in life. This disease followed a sequence of proliferative, membranoproliferative and sclerotic glomerulonephritis. Anti-DNA antibodies were found in the blood of the mice along with immune deposits containing immunoglobulins in subepithelial locations of the glomerular basement membrane. The lesions were unlike those found in aging wild-type B6;129 mice or mice of other strains. Donor found that macrophages from CYP1B1-null mice were impaired in the phagocytosis of apoptotic, necrotic, and opsonized cells. This suggests a generalized defect in the phagocytic activity of CYP1B1-null mouse macrophages. Male mice also developed a high incidence (62-64%) of histiocytic sarcomas. Donor's study provides evidence that deficiency of CYP1B1 can play a role in the development of glomerular disease, normal processing of catabolic DNA and tumors of the mononuclear phagocyte system. The function of CYP1B1 in histiocytes and macrophages may involve both self-tolerance and tumor suppression (Ward JM, Nikolov NP, Tschetter JR, Kopp JB, Gonzalez FJ, Kimura S, Siegel RM. Toxicol Pathol. 2004 Nov-Dec;32(6):710-718).

Heterozygous: Same as wild-type.

The plasmid DNA used for targeting was purified by banding twice on cesium chloride. After linearization by ClaI, 5, 10, 20 or 40

The MMRRC Centers have developed a genetic QC pipeline using MiniMUGA array genotyping to provide additional information on strain backgrounds for MMRRC congenic and inbred strains. For more information on when data may be available, or to request genotyping for a strain of interest, please contact mmrrc@med.unc.edu. Older strains may not have this information.

Frank J. Gonzalez, Ph.D., National Institutes of Health, National Cancer Institute

Ward JM, Nikolov NP, Tschetter JR, Kopp JB, Gonzalez FJ, Kimura S, Siegel RM. Progressive glomerulonephritis and histiocytic sarcoma associated with macrophage functional defects in CYP1B1-deficient mice. Toxicol Pathol. 2004 Nov-Dec;32(6):710-8. (Medline PMID: 15580705)