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

Authors

• S Mahmood
• ABB Ferreira
• CS Lewis
• O Oluwayiose
• S Lu
• J Leung
• J Lack
• LA Martins
• J Kotal
• JM Ribeiro
• L Tirloni

Abstract

Arginine vasopressin (AVP) is a nonapeptide hormone coded by the AVP gene, synthesized in the hypothalamus and secreted by the posterior pituitary. Previous studies of 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 were difficult to breed, and commercial breeders have ceased production. Therefore, the main goal of this project is to create an experimental Avp knockout mouse model that could be used in renal and neuroendocrine research. A mixture of CRISPR elements, including sgRNAs, Cas9 mRNA, ssODN, and loxP sites was injected into C57BL/6 embryos. Successful insertion of the loxP sites was confirmed by PCR using primers flanking the targeted regions. For additional confirmation, sequencing analysis was performed. Mice harboring the floxed allele were mated to mice that globally express a tamoxifen-inducible Cre recombinase. The resultant inducible Avp knockout mice (flox/flox;Cre/wt) show no signs of polydipsia or polyuria prior to induction, indicating that the floxed gene maintains its wild-type function. The administration of an exogenous inducer like tamoxifen to (8-10) week-old mice, induced Cre mediated recombination that resulted in a decrease in urine osmolality. Sanger sequencing demonstrated the expected 1245 bp deletion at the Avp locus. Immunoblotting of AQP2 in the inner medulla showed a significant decrease in AQP2 abundance. This inducible Avp knockout mouse model provides researchers with a valuable tool to investigate the consequences of Avp gene deletion in a controlled and inducible manner.

Scientific Focus Area: Systems Biology

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