Khundmiri, SyedKnepper, MarkKhan, Shaza2023-07-272023-07-272023-07-14https://hdl.handle.net/20.500.14154/68731Background: Arginine vasopressin (AVP) is a nonapeptide 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 were difficult to breed owing largely to the fixed nature of the Avp mutation, resulting in high incidences of fetal and neonatal deaths, behavioral abnormalities in adults, as well as small litter size. 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. Methods: A mixture of CRISPR elements, including active sgRNAs, Cas9 mRNA, single- stranded oligonucleotides (ssODN), and loxP sites was injected into C57BL/6 embryos. 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, the amplified PCR products were subsequently cloned into the TA-cloning vector and subjected to sequencing analysis. 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. Results: 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 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 (flox/flox;Cre/wt) mice to 27 ±1 4 % of values in Cre- floxed control mice. Conclusion: 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. By activating the inducible Cre recombinase at specific developmental stages, researchers can study the effects of Avp deletion on various physiological processes, such as water balance, blood pressure regulation, and social behavior.117en-USAVPCRISPR/Cas9Cre/loxPThesis