Kidney Week

Abstract: FR-PO257

Generation of Mice Expressing an N-Terminal SNAP-Tagged Pkd1 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

• Parnell, Stephen C., University of Kansas Medical Center Department of Internal Medicine, Kansas City, Kansas, United States

Stephen C. Parnell,

• Tran, Pamela Vivian, University of Kansas Medical Center Department of Internal Medicine, Kansas City, Kansas, United States

Pamela Vivian Tran,

• Riddle, Heather A.L., University of Kansas Medical Center Department of Internal Medicine, Kansas City, Kansas, United States

Heather A.L. Riddle,

• Wang, Wei, University of Kansas Medical Center Department of Internal Medicine, Kansas City, Kansas, United States

Wei Wang,

• Haycraft, Courtney J., The University of Alabama at Birmingham Department of Cell Developmental and Integrative Biology, Birmingham, Alabama, United States

Courtney J. Haycraft,

• Outeda, Patricia, University of Maryland School of Medicine, Baltimore, Maryland, United States

Patricia Outeda,

• Yoder, Bradley K., The University of Alabama at Birmingham Department of Cell Developmental and Integrative Biology, Birmingham, Alabama, United States

Bradley K. Yoder,

• Watnick, Terry J., University of Maryland School of Medicine, Baltimore, Maryland, United States

Terry J. Watnick,

• Wallace, Darren P., University of Kansas Medical Center Department of Internal Medicine, Kansas City, Kansas, United States

Darren P. Wallace,

• Calvet, James P., University of Kansas Medical Center Department of Internal Medicine, Kansas City, Kansas, United States

James P. Calvet,

Group or Team Name

• PKD Research Resource Consortium

Background

ADPKD missense mutations that impair intracellular trafficking of polycystin-1 (PC1) indicate that PC1 undergoes complex regulation and interacts with various proteins to mature and reach its cellular destinations. However, a lack of antibodies that reliably detect endogenous PC1 and the inability to analyze PC1 trafficking in live cells have limited our understanding of the interplay between PC1 function and localization. To facilitate the in vivo visualization of PC1 trafficking and co-immunoprecipitation of multiprotein complexes, we have engineered mice that express endogenous PC1 with an N-terminal SNAP tag.

Methods

For proof-of-concept studies, a cDNA construct expressing N- and C-terminally tagged PC1 (^SNAPPC1^CLIP) was transfected into HEK293T cells. ^SNAPPC1^CLIP was detected by Western blot and localized in live cells using SNAP ligand, followed by immunostaining for the ciliary marker, ARL13B. Subsequently, a homologous repair construct encoding SNAP fused in-frame with the first exon of Pkd1 was injected into C57BL/6J pronuclei for homology driven repair using CRISPR technology. PCR and sequencing were used to screen pups for correct insertion of SNAP into the Pkd1 locus. RT-PCR and matings with Pkd1^V and Pkd1^RC hypomorphic mutant mice were used to examine Pkd1^N-SNAP expression and PC1^N-SNAP functionality.

Results

^SNAPPC1^CLIP localized around the nucleus (likely the endoplasmic reticulum) and in the cilium of HEK293T cells, demonstrating that PC1-N-SNAP can exit the Golgi and traffic to the cilium. Pkd1^N-SNAP mosaic founders were produced and a stable line with confirmed integration was identified. Pkd1-N-SNAP mRNA was transcribed in multiple tissues. At 16 weeks of age, Pkd1^N-SNAP/V and Pkd1^N-SNAP/RC compound heterozygotes appeared normal, suggesting that the Pkd1^N-SNAP allele is functional.

Conclusion

Pkd1^N-SNAP mice express the tagged Pkd1 allele and the tag does not appear to impair PC1 function. Future experiments will test SNAP ligands in primary cells and kidneys of live mice to visualize intracellular trafficking of PC1^N-SNAP. These mice should enable novel investigation of the regulation of intracellular trafficking and the cellular functions of PC1.

Funding

• NIDDK Support