B6.FVB-Agtr1a^em1(cre)Zak/J

Stock number: 031487
Product name: Agtr1a-2A-Cre
Research areas: Neurobiology Research, Research Tools

Description

Agtr1a-2A-Cre knock-in mice are designed to have Cre recombinase expression directed to Agtr1a-expressing cells. Cre activity recapitulates that of the endogenous Agtr1a gene in the brain. These mice may be used to generate conditional mutations for studying neural circuits underlying thirst, vasopressin secretion, and fluid homeostasis.

Detailed description

The targeted Agtr1a gene encodes a receptor for the vasopressor hormone angiotensin II and is involved in regulation of blood pressure, blood volume, electrolyte homeostasis. Mutations in this gene have been associated with hypertension and renal tubular dysgenesis. These Agtr1a-2A-Cre mice carry a knock-in mutation that has a T2A sequence and Cre recombinase gene inserted immediately upstream of the Agtr1a STOP codon. cre expression is controlled by the endogenous Agtr1a promoter and regulatory elements. When these mice are bred with mice containing lox-flanked sequences, Cre-mediated recombination will result in deletion of the floxed sequences in Agtr1a-expressing cells in the offspring. Cre recombinase activity is detected in neurons of the forebrain lamina terminalis including the subfornical organ (SFO), organum vasculosum of the lamina terminalis (OVLT), and median preoptic nucleus (MnPO), which are part of the thirst neural circuit and respond to fluid imbalance to regulate drinking behavior. The Donating Investigator did not examine cre expression in tissues other than brain. Homozygotes are viable and fertile.

Development

The bicistronic Agtr1a-2A-Cre knock-in allele was created using homologous recombination (aided by targeted CRISPR/cas9 endonuclease activity) to insert a viral 2A oligopeptide sequence (T2A; mediates ribosomal skipping) and a Cre recombinase gene (cre) just upstream of the endogenous STOP codon of the Agtr1a gene. The targeting vector, Cas9 endonuclease and single guide RNA were mixed and injected into the pronuclei of FVB/N fertilized zygotes. Embryos were transferred to pseudopregnant females, and correctly targeted pups (identified by PCR and sequencing) were bred to C57BL/6J inbred mice for germline transmission. The mice were then backcrossed onto C57BL/6J for four generations. Upon arrival, sperm was cryopreserved. To establish our live colony, an aliquot of frozen sperm was used to fertilize C57BL/6J oocytes (Stock No. 000664).

Allele

Symbol: Agtr1a^em1(cre)Zak
Name: endonuclease-mediated mutation 1, Zachary Knight
MGI accession ID: MGI:6117421
Generation method: Endonuclease-mediated
Attributes: Recombinase-expressing
Marker symbol: Agtr1a
Marker name: angiotensin II receptor, type 1a
Chromosome: 13
Strain of origin: FVB/N
Expressed genes: cre,cre recombinase,bacteriophage P1
Site of expression: When these mice are bred with mice containing lox-flanked sequences, Cre-mediated recombination will result in deletion of the floxed sequences in Agtr1a-expressing cells in the offspring. Cre recombinase activity is detected in neurons of the forebrain lamina terminalis including the subfornical organ (SFO), organum vasculosum of the lamina terminalis (OVLT), and median preoptic nucleus (MnPO).
Molecular note: The bicistronic Agtr1a-2A-Cre knock-in allele was created using homologous recombination (aided by targeted CRISPR/cas9 endonuclease activity) to insert a viral 2A oligopeptide sequence (T2A; mediates ribosomal skipping) and a Cre recombinase gene (cre) immediately upstream of the endogenous STOP codon of Agtr1a gene. The targeting vector, Cas9 endonuclease and single guide RNA were mixed and injected into the pronuclei of FVB/N fertilized zygotes. Embryos were transferred to pseudopregnant females, and correctly targeted pups were maintained on mixed FVB/C57BL/6J background. Two independent knock-in lines were crossed to reporter mice, and reporter expression patterns from these lines were identical.