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Kidney Week

Abstract: FR-PO743

A Novel CRISPR/Cas9 eGFP Knockin Mouse for Characterizing Endogenous Polycystin 1

Session Information

Category: Genetic Diseases of the Kidneys

  • 1001 Genetic Diseases of the Kidneys: Cystic

Authors

  • Lin, Cheng-Chao, National Institutes of Health, Bethesda, Maryland, United States
  • Menezes, Luis F., National Institutes of Health, Bethesda, Maryland, United States
  • Zhou, Fang, National Institutes of Health, Bethesda, Maryland, United States
  • Germino, Gregory G., National Institutes of Health, Bethesda, Maryland, United States
Background

Mutations in Pkd1 are identified in the majority of cases of autosomal dominant polycystic kidney disease (ADPKD). This gene encodes a large trans-membrane protein, Polycystin 1 (PC1), which undergoes complex processing that results in multiple cleavage products. The complexity and low abundance of PC1 makes investigation into the functions of endogenous protein extremely challenging, and most of our knowledge of PC1 comes from study of recombinant protein over-expressed in heterologous systems. In order to investigate PC1 function under physiological conditions, we generated a knock-in mouse model that expresses a chimeric PC1-eGFP-3HA fusion protein under the control of its native promoter using the CRISPR/Cas9 method.

Methods

We characterized the expression of PC1-eGFP-3HA by immunoblot (IB), immunoprecipitation(IP), and immunofluorescence(IF) in mouse tissues and in Murine Embryonic Fibroblasts (MEFs) and Renal Epithelial Cells (REC) derived from the mouse line. ThePkd1-eGFP-3HA/Pkd1 and Pkd1/KO mouse lines were intercrossed to test if the fusion protein was functional.

Results

Genomic PCR and sequencing results confirmed the successful insertion of eGFP and 3HA tag sequences into the Pkd1 locus. Offspring of Pkd1-eGFP-3HA/Pkd1 X Pkd1/KO crosses were obtained at mendelian ratios. Neither the kidneys nor livers of Pkd1-eGFP-3HA homozygotes were cystic at 1 year, and they remain healthy at >1.5 years without other apparent abnormality. The PC1-eGFP-3HA fusion protein can be reliably detected by IB and IP in various tissues and cell lines. Multiple previously reported PC1 cleavage products were detected in a variety of tissues. However, we have not yet been successful in detecting PC1-specific signal above background by IF in any tissues. Live cell imaging and IF in MEF cells using various antibody, fixation and microscopy methods failed to detect PC1. So far, we can only visualize the fusion protein unambiguously by IF in primary cilia of REC.

Conclusion

The PC1 fusion protein function appears to be as functional as untagged protein and could be detected more easily and reliably. Using GFP-nanobody magnetic beads, the affinity purification–mass spectrometry (AP-MS) for endogenous PC1 fusion protein is also more efficient. This model will be a helpful resource for studying endogenous PC1 trafficking in live cells and how it interacts with other proteins.

Funding

  • NIDDK Support