Replacing the Brattleboro Rat: Generation of an Inducible Avp Knockout Mouse Line Using CRISPR/Cas9 and Cre/LoxP

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Background: Arginine vasopressin (AVP) is a peptide hormone coded by the Avp gene, synthesized in the hypothalamus and secreted by the posterior pituitary. Dysregulation of AVP secretion contributes to a variety of human diseases. Previous studies of functional roles of AVP have been largely dependent on the use of Brattleboro rats, which manifest a spontaneous mutation in the Avp gene and lack circulating AVP. Despite their utility, Brattleboro rats are difficult to breed owing largely to the fixed nature of the Avp mutation, resulting in increased neonatal death and behavioral effects in the adults. Consequently, commercial breeders have ceased production, despite a continued need. To fill this need, we have now created a mouse model with tamoxifen-inducible deletion of AVP. Methods: We employed CRISPR/Cas9 to flox a portion of exon 2 of the Avp gene. Successful insertion of the two loxP sites was confirmed by PCR using primers flanking the targeted regions. Restriction enzymes BamHI or EcoRI were used to confirm correct targeting. For additional confirmation, PCR products were cloned into the TA-cloning vector and sequenced. Mice harboring the floxed allele were mated to mice that globally express a tamoxifen-inducible Cre recombinase. Results: The resultant homozygous conditional knockout mice (Cre+Avpflx/flx) are viable, fertile and show no signs of polydipsia or polyuria prior to induction, indicating that the conditional alleles retain their wild-type function. Induction of Cre-mediated recombination by administration of tamoxifen (2-3 mg q.d., i.p. for 5 days) in 8-week-old mice resulted in a decrease in urine osmolality from 2076±138 to 122±6 mOsm/kgH2O on day 31 after induction (P<0.0001). Sanger sequencing demonstrated the expected 1245 bp deletion at the Avp locus. The kidney parenchyma was structurally normal with no evidence of medullary atrophy 6 weeks after induction. Immunoblotting of AQP2 in the inner medulla showed a significant decrease in AQP2 band density in Cre+Avpflx/flx mice to 27±14% of values in Cre- floxed control mice (P<0.05). Conclusions: We developed an inducible AVP knockout mouse line Cre+Avpflx/flx that will be shared with the research community and are likely to be useful for further study of regulation of water balance and polycystic kidney disease, as well as neural, vascular, and metabolic roles of AVP. Funding: Other NIH Support - NHLBI

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