Inducible Avp Knockout Mouse Line

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. Therefore, the main goal of this project is to create an effective experimental Avp knockout mouse model that could be used in renal and neuroendocrine research to study the control of water balance by AVP. 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. Mice harboring the floxed allele were mated to B6.Cg-Tg(CAG-cre/Esr1*)5Amc/J mice that globally express a tamoxifen-inducible Cre recombinase. The resultant inducible Avp knockout mice (Cre+Avpflx/flx) 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 from 2076 ± 138 to 122 ± 6 mOsm/kgH2O on day 31 after induction. 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 band density in (Cre+Avpflx/flx) mice to 27 ±1 4 % of values in Cre- floxed control mice. 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. Overall design: The process begins with the design (CHOPCHOP) and synthesis of guide RNAs (gRNAs) that target the desired regions (first intron and 3' downstream) within the vasopressin gene. These gRNAs, when complexed with the Cas9 nuclease, induce site-specific double-strand breaks (DSBs) at the AVP locus. To introduce the loxP sites, a DNA repair template containing the loxP sequences and restriction enzyme sites (BamHI and EcorI) are provided along with the CRISPR/Cas9 components. The repair template serves as a template for homology-directed repair (HDR) and facilitates the precise insertion of loxP sequences flanking the vasopressin gene. Successful insertion of the two loxP sites was confirmed by PCR using primers flanking the targeted regions. Restriction enzymes were used to confirm correct targeting. Additionally, PCR amplification of DNA sequences spanning the two loxP sites was carried out, followed by cloning into the TA-cloning vector and subsequent sanger sequencing for further confirmation. Sequencing analysis of the founder mice demonstrated precise insertion of the loxP sites into each intended location within the Avp locus, without introducing mutations at the loxP sites.