Kidney Week

Abstract: FR-PO595

Functional Studies of Polycystin-1 Using a Novel Pkd1-HaloTag Mouse

Session Information

• Cystic Kidney Diseases: Mechanisms and Models
  October 25, 2024 | Location: Exhibit Hall, Convention Center
  Abstract Time: 10:00 AM - 12:00 PM

Category: Genetic Diseases of the Kidneys

• 1201 Genetic Diseases of the Kidneys: Cystic

Authors

• Cordido, Adrian, Yale University, New Haven, Connecticut, United States

Adrian Cordido, PhD

• Dong, Ke, Yale University, New Haven, Connecticut, United States

Ke Dong, MD

• Cai, Yiqiang, Yale University, New Haven, Connecticut, United States

Yiqiang Cai, MD, PhD

• Tian, Xin, Yale University, New Haven, Connecticut, United States

Xin Tian, MD

• Wei, Zemeng, Yale University, New Haven, Connecticut, United States

Zemeng Wei

• Rehman, Michael, Yale University, New Haven, Connecticut, United States

Michael Rehman, PhD

• Somlo, Stefan, Yale University, New Haven, Connecticut, United States

Stefan Somlo, MD

Background

Autosomal dominant polycystic kidney disease (ADPKD) is a common genetic disease caused primarily by mutations in genes encoding Polycystin-1 (PC1) and Polycystin-2 (PC2). The physiological function of polycystins and the process through which their dysfunction leads to cystogenesis remains unclear. The localized expression of PCs, particularly PC1, in kidney and other tissues has been challenging to detect. HaloTag is a versatile protein labeling system which allows antibody independent covalent ligand-based labeling of the targeted protein and can improve the sensitivity and intensity of the protein localization signals. We applied this system to PC1 in vivo.

Methods

Based in our preliminary data in transfected cells showing that PC1-HaloTag expression can be detected by HaloTag ligand labeling in western blotting and live cell immunofluorescence, we produced a Pkd1-knockin mouse with a C-terminal fusion of 3XHA-HaloTag with PC1 (PC1^Halo). We characterized the viability and kidney phenotype of the mice and the maturation and expression of PC1^Halo by western blotting and confocal immunofluorescence microscopy.

Results

Pkd1^Halo/Halo mice are born in normal Mendelian ratios and have normal renal and liver phenotypes at 6 months age indicating that PC1^Halo is fully functional in vivo. Adult induced Pkd1^fl/Halo; Pax8^rtTA; TetO-Cre mice are also normal. We detected normal maturation of PC1^Halo by western blot of multiple tissue lysates and primary kidney cell cultures from Pkd1^Halo/Halo mice. We detected endogenous PC1^Halo in cilia of primary kidney cell culture from Pkd1^Halo/Halo mice by confocal imaging following HaloTag ligand labeling in live cells as well as fixed cells.

Conclusion

PC1 C-terminal fusion with HaloTag produces a fully functional PC1^Halo in vivo. Endogenous PC1^Halo can be detected in tissue lysates and can be imaged in primary kidney cell cultures. PC1^Halo will be a useful tool for exploring native PC1 in vitro and in vivo.