STOCK Cyp1a^tm1Dwn Tg(CYP1A1,CYP1A2)1Dwn/J

Stock number: 007580
Research areas: Cancer Research, Cell Biology Research, Research Tools

Description

These humanized hCYP1A1_1A2_Cyp1a2/Cyp1a1(-/-) mice may 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 cytochrome P450 family members.

Detailed description

Mice homozygous for the Cyp1a2/Cyp1a1(-) targeted 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 expression of either gene transcript or protein is observed in liver or small intestine by quantitative RT-PCR or Western blot, respectively. Transgene expression of the orthologous human genes is observed in the same tissues. While oral benzo[alpha]pyrene (BaP) treatment of Cyp1a2/Cyp1a1(-/-) mutant mice leads to BaP-induced immunosuppression and sickness, the presence of the human CYP1A orthologs in these mice minimizes/prevents such toxicity. These humanized hCYP1A1_1A2_Cyp1a2/Cyp1a1(-/-) mice may 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 cytochrome P450 family members.

Development

"Humanized" hCYP1A1_1A2_Cyp1a1/1a2(-/-) mice have interchromosal deletion of a segment of mouse chromosome 9 including the majority of the closely-linked Cyp1a2 and Cyp1a1 genes and also harbor a human CYP1A2 and CYP1A1transgene. Generation of these mice required multiple combinations of other targeting strategies. In detail, the Cyp1a2(t) targeted allele was first 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). Following electroporation into 129S6/SvEvTac-derived embryonic stem (ES) cells and ES cell microinjection into C57BL/6 blastocysts, the chimera's were bred with C57BL/6 to generate Cyp1a2(t) mutant mice. To create the Cyp1a1 targeted allele, 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 129P2/OlaHsd-derived E14TG2a (HPRT-) ES cells, ES cells were microinjected into the blastocoele cavity of C57BL/6J embryos, and chimeric males were bred with C57BL/6J females. These Cyp1a1(t) mutant mice were then bred to a Cre-recombinase strain (CAGGS-CRE, mixed C57BL/6J and FVB/J genetic background). Transgenic offspring 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) and hemizygous for CAGGS-CRE were bred to mice heterozygous for the Cyp1a2(t) allele. Mutant mice (Cyp1a2(t), Cyp1a1(-), CAGGS-CRE) were bred to C57BL/6. Because of the close genomic position of these two genes, offspring could be selected which had undergone Cre recombinase-mediated interchromosomal recombination between the loxP sites 3' beyond the stop codons of the Cyp1a2 and Cyp1a1 genes. Such mice were backcrossed to C57BL/6 for 10 generations (while selecting against the Cre transgene) to generate Cyp1a1/1a2 mutant mice. 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/6JxDBA/2J)F1 oocytes. Founder mice having a single copy of BAC-H were identified and then backcrossed for 10 generations to C57BL/6 mice. These hCYP1A1_1A2 transgenic mice were next bred with Cyp1a1/1a2 mutant mice, generating the final mutant strain; "humanized" hCYP1A1_1A2_Cyp1a1/1a2(-/-) mice.

NOTE: A 22 SNP (single nucleotide polymorphism) panel analysis performed by The Jackson Laboratory revealed that this strain is on a mixed STOCK background (5 out of 22 markers are segregating for C57BL/6, FVB/N, or 129).

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: 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.