Kidney Week

Abstract: FR-PO545

Addition of an N-Terminal SNAP-Tag Induces a Novel Hypomorphic Pkd1 Allele

Session Information

• Genetic Diseases: Cystic - Basic
  November 03, 2023 | Location: Exhibit Hall, Pennsylvania Convention Center
  Abstract Time: 10:00 AM - 12:00 PM

Category: Genetic Diseases of the Kidneys

• 1201 Genetic Diseases of the Kidneys: Cystic

Authors

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

Stephen C. Parnell,

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

Pamela Vivian Tran,

• Prasad, Gauri, University of Kansas Medical Center, Kansas City, Kansas, United States

Gauri Prasad,

• Unger, Jordan A., University of Kansas Medical Center, Kansas City, Kansas, United States

Jordan A. Unger,

• Haycraft, Courtney J., University of Alabama, Birmingham, Alabama, United States

Courtney J. Haycraft,

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

Patricia Outeda,

• Yoder, Bradley K., University of Alabama, 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, Kansas City, Kansas, United States

Darren P. Wallace,

Group or Team Name

• On Behalf of the Polycystic Kidney Disease Research Resource Consortium (PKD-RRC).

Background

We engineered mice that express endogenous polycystin-1 (PC1) with an N-terminal, self-labeling SNAP-tag to facilitate the in vivo visualization of PC1 trafficking and co-immunoprecipitation of multiprotein complexes. While initial reports indicated that the allele is normal, follow-on work has demonstrated that addition of SNAP results in a hypomorphic cystic phenotype that is variable depending on the nature of the second Pkd1 allele.

Methods

A repair construct encoding SNAP fused in-frame with the first exon of Pkd1 was injected into C57 pronuclei for homology-driven repair using CRISPR. Resulting Pkd1^N-SNAP mice were crossed to homozygosity and also crossed with non-functional Pkd1^NdL (truncation) mice, and hypomorphic Pkd1^V and Pkd1^RC mice, to generate compound heterozygotes. Mice were maintained up to 26-weeks of age, then sacrificed for analysis.

Results

Compound heterozygous Pkd1^N-SNAP/NdL pups had severely cystic kidneys at 2 weeks of age, with an average percent kidney weight to body weight (%KW/BW) of 9.4%. Homozygous Pkd1^{N-SNAP/N-SNAP} mice had moderately cystic kidneys with a %KW/BW of 2.6% at 8 weeks, progressing to 5.2% at 23-26 weeks. In contrast to compound heterozygous Pkd1^V/RC mice, which are severely cystic by 3 weeks, compound heterozygous Pkd1^N-SNAP/V and Pkd1^N-SNAP/RC mice had kidneys with only a few very small cysts with a %KW/BW of 1.4% at 23-26 weeks. Interestingly, a female Pkd1^N-SNAP/V mouse and a female Pkd1^N-SNAP/RC mouse used as breeders had a few large cysts at 23-26 weeks.

Conclusion

Addition of SNAP to the N-terminus of PC1 generates a hypomorphic Pkd1^N-SNAP allele. The cystic phenotypes in Pkd1^N-SNAP/NdL and Pkd1^{N-SNAP/N-SNAP} mice suggest that the tag affects PC1 trafficking, stability, and/or a critical function of the polycystin complex. However, the very mild phenotype in Pkd1^N-SNAP/V and Pkd1^N-SNAP/RC mice suggests complementation between the SNAP allele and the V or RC alleles. This suggests the PC1-SNAP defect is functionally distinct from that of the defects caused by the V and RC mutations, allowing partial preservation of PC1 function when present in trans within the same animal. Future experiments will determine the functionality of the SNAP-tag, the localization of the PC1^N-SNAP protein, and the nature of the defect caused by the SNAP-tag.

Funding

• NIDDK Support