Abstract: FR-PO252
Tracking Polycystin2 In Vivo Using a Novel Engineered Pkd2-HALO Allele
Session Information
- Genetic Diseases of the Kidneys: Cystic - II
November 04, 2022 | Location: Exhibit Hall, Orange County Convention Center‚ West Building
Abstract Time: 10:00 AM - 12:00 PM
Category: Genetic Diseases of the Kidneys
- 1101 Genetic Diseases of the Kidneys: Cystic
Authors
- Haycraft, Courtney J., The University of Alabama at Birmingham School of Medicine, Birmingham, Alabama, United States
- Croyle, Mandy J., The University of Alabama at Birmingham School of Medicine, Birmingham, Alabama, United States
- Lambert, Laura, The University of Alabama at Birmingham School of Medicine, Birmingham, Alabama, United States
- Outeda, Patricia, University of Maryland School of Medicine, Baltimore, Maryland, United States
- Xu, Hangxue, The University of Alabama at Birmingham School of Medicine, Birmingham, Alabama, United States
- Zhou, Juling, The University of Alabama at Birmingham School of Medicine, Birmingham, Alabama, United States
- Chumley, Phillip H., The University of Alabama at Birmingham School of Medicine, Birmingham, Alabama, United States
- Clearman, Kelsey R., The University of Alabama at Birmingham School of Medicine, Birmingham, Alabama, United States
- Qian, Feng, University of Maryland School of Medicine, Baltimore, Maryland, United States
- Mrug, Michal, The University of Alabama at Birmingham School of Medicine, Birmingham, Alabama, United States
- Tran, Pamela Vivian, University of Kansas School of Medicine, Kansas City, Kansas, United States
- Parnell, Stephen C., University of Kansas School of Medicine, Kansas City, Kansas, United States
- Watnick, Terry J., University of Maryland School of Medicine, Baltimore, Maryland, United States
- Yoder, Bradley K., The University of Alabama at Birmingham School of Medicine, Birmingham, Alabama, United States
Group or Team Name
- PKD Research Resource Consortium
Background
ADPKD, one of the leading causes of end stage renal disease, is due to mutations in the genes PKD1 or PKD2, which encode for polycystin1 or polycystin2, respectively. While the genes involved were identified several decades ago, research is ongoing to understand their functions and how disruption leads to renal cyst development. Studies focused on trafficking and localization of polycystin2 (PC2) are hindered by the difficulty in antibody generation, low-level expression of the endogenous protein, and inability to track the protein in live cells.
Methods
To expand the reagents available to the PKD research community and accelerate studies of PC2 function, the PKD RRC used CRIPSR/Cas9 to target HALO to the C-terminus of the endogenous mouse Pkd2 gene.
Results
Mice homozygous for the engineered Pkd2-HALO allele are viable and have no overt renal cysts at three months of age indicating that the PC2-HALO protein is functional. Initial studies using western blot from tissues harvested from heterozygous mice showed expression of the PC2-HALO protein in brain and kidney lysates. Additionally, we have begun testing the functionality of the HALO ligand in vivo and in primary cells cultured from the mouse line. We confirmed, using a HALO-ligand in heterozygous primary cultured cells, the presence of fluorescent labeling of the fusion protein in the primary cilium of the renal epithelium. Additional characterization of this novel mouse resource is ongoing.
Conclusion
This mouse will provide an invaluable resource to analyze PC2 function, trafficking, and turnover in vivo.
Funding
- NIDDK Support