B6.129(Cg)-Ahr^em1Bra/J

Stock number: 035734
Product name: Ahr^fx Ahr^V375A
Research areas: Cancer Research, Cardiovascular Research, Cell Biology Research, Developmental Biology Research, Research Tools

Description

The Ahr^V375A Ahr^fx strain provides a high affinity receptor conditional model useful in generating tissue-specific mutants of the exon 2 floxed allele of the aryl-hydrocarbon receptor (Ahr) for use in toxicology and drug studies involving the AHR pathway and fetal vascular development such as ductus venosus closure.

Of note, the low affinity conditional Ahr allele, Ahr^tm3.1Bra, is available as Stock No. 006203.

Detailed description

Ahr (aryl hydrocarbon receptor) is a ligand activated HLH (helix loop helix) transcription factor and is part of the PER-ARNT-SIM (PAS) environmental sensor superfamily involved in xenobiotic metabolism, toxicity of environmental pollutants, vascular development and immune function. The Ahr^V375A allele is a valine to alanine amino acid substitution at codon 375 (V375A) in the Ahr ligand binding domain; the mutation converts a low affinity receptor into a high affinity receptor. A second mutation in Ahr, the conditional Ahr^fx allele carries a loxP sites on either side of exon 2. Double homozygous mutants are viable and fertile. Following administration of an Ahr agonist, hepatic mRNA transcript levels of AHR-driven genes are between 2.6-9.6 fold higher in homozygous Ahr^V375A mice than in mice carrying the low affinity Ahr^d allele. When bred to mice that express tissue-specific Cre recombinase, resulting offspring will have the exon 2 deleted in cre-expressing tissues.
For example, when bred to mice with endothelial cell specific (Tg(Cdh5-cre)7Mlia, Stock No. 006137) Cre recombinase expression, homozygous knockout offspring exhibit patent ductus venosus in 100% of mice.

Of note, the low affinity conditional Ahr allele, Ahr^tm3.1Bra, is available as Stock No. 006203.

Development

The Ahr^fx targeting vector was designed to place a loxP-flanked PGK-neo cassette immediately upstream of exon 2 of the endogenous gene, as well as insert a third loxP site downstream of exon 2. The targeting vector was built from a clone containing a 15 kb region of homology to the murine Ahr locus from a 129SvJ genomic library (129SvJ carries the Ahr^d allele of the Ahr locus). The construct was electroporated into 129SvJ-derived GS1 embryonic stem (ES) cells (Genome Systems, St. Louis). Correctly targeted ES cells were injected into recipient blastocysts. Chimeric mice were bred to FVB/N-Tg(EIIa-cre)C5379Lmgd/J (Stock 003314) mice to remove the upstream PGK-neo cassette. After confirmation of PGK-neo excision, mice carrying loxP-flanked exon 2 were then bred to C57BL/6J-background mice to remove the EIIa-Cre transgene and produce the parental line (Ahr^fx). Because the conditional Ahr^fx allele was generated originally from 129SvJ genome (targeting vector and ES cells) that carry the lower affinity Ahr^d allele, the donating investigator used a C57BL/6J strain congenic for DBA2-derived Ahr^d allele (B6.D2N-Ahr^d/J Stock No. 002921) to perform all backcrosses prior to generating mice homozygous for the Ahr^fx mutation.

For the Ahr^V375A targeting vector, CRISPR/Cas9 genome editing was used to introduce a valine to alanine amino acid substitution at codon 375 (GAC to GGC, V375A) and 7 bases downstream a silent mutation designed to disrupt a targeted cut site to prevent subsequent cleavage and indel formation. The mutation is located in the ligand binding domain of Ahr. Single guide RNA and Cas9 endonuclease were introduced into fertilized eggs derived from mice carrying the Ahr^fx (Ahr^tm3.1Bra, see Stock No. 006203) mutation on a congenic C57BL/6J background by pronuclear injection. Embryos were transferred to pseudopregnant females, and correctly targeted pups were bred to C57BL/6J mice for germline transmission. The V375A mutation was confirmed by sequencing. The colony has been backcrossed to C57BL/6 for at least 5 generations. Upon arrival, sperm was cryopreserved. To establish our live colony, an aliquot of frozen sperm was used to fertilize C57BL/6J oocytes.

Allele

Symbol: Ahr^em1Bra
Name: endonuclease-mediated mutation 1, Christopher A Bradfield
MGI accession ID: MGI:6402393
Generation method: Endonuclease-mediated
Attributes: Not Specified
Synonyms: Ahr^V375A
Marker symbol: Ahr
Marker name: aryl-hydrocarbon receptor
Marker synonyms: Ah; Ahh; Ahre; bHLHe76; dioxin receptor; FVH3; In; RP85
Chromosome: 12
Strain of origin: B6.129(FVB)-Ahr^tm3.1Bra/Bra
Molecular note: CRISPR/Cas9-targeting in Ahr^tm3.1Bra zygotes induced a single nucleotide change. Residue 375 was changed from a valine to an alanine (p.V375A) to change the low-affinity d allele (as found in the 129 strain) to the high-affinity b allele (as found i the B6 strain). There is also a silent mutation downstream. The genetic code (with the mutation denoted by upper case) reads: gGcgatgatAtaat which was changed from gAcgatgatGtaat.