B6.Cg-Cyp1^tm1Dwn Ahr^d Tg(CYP1A1,CYP1A2)1Dwn/DwnJ

Stock number: 008599
Strain synonyms: humanized hCYP1A1_1A2_Cyp1a1/1a2(-/-)_Ahr<d> mutant mice
Research areas: Cancer Research, Cell Biology Research, Metabolism Research, Research Tools

Description

These "humanized" hCYP1A1_1A2_Cyp1a1/1a2(-/-)_Ahr^d mice carry the human CYP1A1 and CYP1A2 genes in the absence of functional mouse Cyp1a1 and Cyp1a2 orthologs, and also mimic the human poor-affinity aryl hydrocarbon receptor (AHR) by carrying the poor-affinity Ahr^d allele derived from DBA/2J mice. These may be useful in drug or carcinogen metabolism research; specifically as a model for human risk assessment studies involving drug or environmental toxicants that may be substrates for the aryl hydrocarbon receptor (AHR) or cytochrome P450 family 1 members.

Detailed description

These "humanized" hCYP1A1_1A2_Cyp1a1/1a2(-/-)_Ahr^d mice carry the human CYP1A1 and CYP1A2 genes in the absence of functional mouse Cyp1a1 and Cyp1a2 orthologs, and also mimic the human poor-affinity aryl hydrocarbon receptor (AHR) by carrying the poor-affinity Ahr^d allele derived from DBA/2J mice (rather than the high-affinity Ahr^b1 allele normally present on a C57BL/6J genetic background); all on a C57BL/6J (reported >99.8%) genetic background.

Mice homozygous for the Cyp1a2/Cyp1a1 targeted allele [Cyp1a1/1a2(-/-)], homozygous for the Ahr^d allele, and carrying the hCYP1A1_1A2 transgene are viable and fertile with normal lifespan. As the Cyp1a2/Cyp1a1(-) targeted allele lacks the coding regions of both Cyp1a1 and Cyp1a2 genes, no mouse CYP1A1 or CYP1A2 mRNA expression is observed in liver, lung or kidney. Transgene expression of the orthologous human genes is observed in the same tissues. Because expression of the hCYP1A1_1A2 transgene is controlled by the mouse AHR, the low-affinity Ahr^d allele results in diminished CYP1A responses following exposure to AHR inducers (such as dioxin) when compared to hCYP1A1_1A2_Cyp1a1/1a2(-/-) mice harboring a high-affinity Ahr allele. These humanized hCYP1A1_1A2_Cyp1a2/Cyp1a1(-/-)_Ahr^d mice may be useful in drug or carcinogen metabolism research; specifically as a model for human risk assessment studies involving drug or environmental toxicants that may be substrates for the aryl hydrocarbon receptor (AHR) or cytochrome P450 family 1 members.

Development

These "humanized" hCYP1A1_1A2_Cyp1a1/1a2(-/-)_Ahr^d mutant mice were generated in the laboratory of Dr. Daniel W. Nebert (University of Cincinnati Medical Center). These mice were generated by breeding hCYP1A1_1A2_Cyp1a1/1a2(-/-) mice (see Stock No. 007580, but reportedly maintained on a >99.8% C57BL/6J genetic background) to B6.D2-Ahr^d mice (Stock No. 002921). The resulting mice were subsequently bred with B6.D2-Ahr^d mice for at least 2 generations to fix the Ahr^d allele to homozygosity. Mice homozygous for the Cyp1a1/1a2 mutation, homozygous for the Ahr^d allele, and carrying the hCYP1A1_1A2 transgene were sent to The Jackson Laboratory. Upon arrival, mice were bred with B6.D2-Ahr^d mice (Stock No. 002921) for at least one generation to establish this colony.

Upon rederivation of these mice at The Jackson Laboratory, we will perform a genetic background analysis using a single nucleotide polymorphism (SNP) analysis. The results of this analysis have not yet been completed (December 2008).

Allele

Symbol: Tg(CYP1A1,CYP1A2)1Dwn
Name: transgene insertion 1, Daniel W Nebert
MGI accession ID: MGI:3721236
Generation method: Transgenic
Attributes: Inserted expressed sequence; Humanized sequence
Marker symbol: Tg(CYP1A1,CYP1A2)1Dwn
Marker name: transgene insertion 1, Daniel W Nebert
Chromosome: UN
Strain of origin: (C57BL/6J x DBA/2J)F1
Expressed genes: CYP1A2,cytochrome P450 family 1 subfamily A member 2,human; CYP1A1,cytochrome P450 family 1 subfamily A member 1,human
Molecular note: To create "humanized" CYP1A1 and CYP1A2 transgenic mice (hCYP1A1_1A2), a single copy of the 180-kb human CYP1A1_CYP1A2 locus-containing BAC-H (BAC Human CTB clone 31H21: including the 23.3 kb spacer region, 90 kb of CYP1A2 3'-flanking region and 53 kb of CYP1A1 3' flanking region) was microinjected into (C57BL/6J x DBA/2J)F1 oocytes. Founder mice having a single copy of BAC-H were identified and then backcrossed for 10 generations to C57BL/6 mice.

Allele

Symbol: Ahr^d
Name: d variant
MGI accession ID: MGI:2667196
Generation method: Not Applicable
Marker symbol: Ahr
Marker name: aryl-hydrocarbon receptor
Chromosome: 12
Strain of origin: Not Applicable
Molecular note: This allele encodes a 104 kDa receptor that is stabilized by molybdate and has an affinity for ligand 10-100 fold lower than that of the receptor produced by the C57BL/6J allele. PCR sequencing of cDNA revealed ten nucleotide differences between the coding sequences of the DBA/2J and C57BL/6J receptors. Five of the ten differences would cause amino acid changes. One of these, an apparent T to C transition replaces the opal termination codon in the C57BL/6J allele with an arginine codon in the DBA/2J allele. This change would extend translation of the DBA/2J mRNA by 43 amino acids, accounting for the larger size of the peptide produced by this allele (104 kDa vs 95 kDa for the C57BL/6J allele). A second T to C transition changes a leucine codon in the C57BL/6J allele to a proline codon in the DBA/2J allele, and would likely change secondary structure of the peptide and thus ligand affinity.
General note:

Strain of origin - this allele was found in DBA/2J, AKR/J, 129, SWR, RF, NZB strains

Allele

Symbol: Cyp1a^tm1Dwn
Name: targeted mutation 1, Daniel W Nebert
MGI accession ID: MGI:3721229
Generation method: Targeted
Attributes: Null/Knockout
Marker symbol: Del(9Cyp1a2-Cyp1a1)1Dwn
Marker name: deletion, Chr 9, Daniel W Nebert 1
Chromosome: 9
Strain of origin: 129P2/OlaHsd or 129S6/SvEvTac
Molecular note: The Cyp1a2(t) targeted allele (J:122409) was generated using a targeting vector designed to insert a loxP-flanked PGK-NEO cassette 350 bp downstream of the endogenous stop codon (about 60 bp 3' of exon 7). The construct was electroporated into 129S6/SvEvTac-derived embryonic stem (ES) cells which were microinjection into C57BL/6 blastocysts. The chimeras were bred with C57BL/6 to generate Cyp1a2(t) mutant mice. To create the Cyp1a1 targeted allele (J:59398), a targeting vector was designed to insert a loxP-flanked hypoxanthine phosphoribosyltransferase (HPRT) minigene in intron 1 and a loxP site downstream of the termination codon in exon 7. Following electroporation into E14TG2a (HPRT-) ES cells and ES cell microinjection into the blastocoele cavity of C57BL/6J embryos, chimeric males were bred with C57BL/6J females. As described in J:86748, these Cyp1a1(t) mutant mice were then bred to a Cre-deleter strain (CAGGS-CRE, mixed C57BL/6J and FVB/NJ genetic background). The resulting transgenic mice found to be heterozygous for the floxed null Cyp1a1(-) allele (containing only exon 1, a portion of intron 1, and one remaining loxP site in the 3' UTR) were bred to mice heterozygous for the Cyp1a2(t) allele (as described in J:122409). Mutant mice (Cyp1a2(t), Cyp1a1(-), CAGGS-CRE) were bred to C57BL/6. Because of the close genomic position of these two genes, offspring having undergone Cre recombinase-mediated interchromosomal recombination between the loxP sites 3' beyond the stop codons of the Cyp1a2 and Cyp1a1 genes could be selected. Such mice were backcrossed to C57BL/6 for 10 generations (while selecting against the Cre-deleter transgene) to generate Cyp1a1/1a2 mutant mice.