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Background <p>Autosomal dominant polycystic kidney disease (ADPKD), primarily driven by <i>PKD1</i> or <i>PKD2</i> mutations, is a prevalent hereditary nephropathy for which large-animal models capturing human renal anatomy and disease tempo remain urgently needed. This study aimed to establish a site-specific human <i>PKD2</i> (<i>hPKD2</i>) transgenic porcine model as a versatile platform capable of supporting two temporally distinct applications: longitudinal evaluation of whether sustained <i>PKD2</i> overexpression is sufficient to induce renal pathology, and—should overexpression prove phenotypically silent—future crossbreeding-based functional rescue of PKD2-knockout lines.</p> Methods and Results <p>A CRISPR/Cas9-mediated, homology-recombination-independent strategy was employed to target full-length <i>hPKD2</i> cDNA into the porcine pH11 safe harbor locus. Somatic cell nuclear transfer yielded five F0 transgenic founders, and natural mating of two founders with wild-type sows produced four F1 transgenic offspring, confirming stable germline transmission. Quantitative real-time PCR and whole-genome sequencing validated single-copy, site-specific transgene integration at the designated locus. Robust <i>hPKD2</i> mRNA and FLAG-tagged polycystin-2 (PC-2) expression were detected in renal and other tissues across both generations. During the initial 12-month monitoring period, serum blood urea nitrogen and creatinine levels remained within normal ranges and no gross histological abnormalities were evident. However, given that these biomarkers are insensitive to early-stage renal impairment, extended observation with more comprehensive phenotyping is required before definitive conclusions regarding renal function can be drawn.</p> Conclusions <p>A transgenic porcine model with stable, single-copy <i>hPKD2</i> integration at the <i>pH11</i> safe harbor locus was successfully generated and shown to permit germline transmission. This platform provides a foundation for long-term investigation of <i>PKD2</i> overexpression pathophysiology and, alternatively, for functional complementation of PKD2-deficient models, thereby advancing both mechanistic and translational ADPKD research.</p>

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Generation of transgenic pigs with targeted insertion of a wildtype copy of human PKD2 gene

  • 期刊论文

  • 发表日期：

    2026年6月23日

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  • Xue-Lin Zhang,
  • Jia-Ning Luo,
  • Yue Cao,
  • Jiu-Xiao Zhao,
  • Yuan Zhang,
  • Sai-Fei Xu,
  • Le-Nan Zhuang,
  • Jin He

摘要

Background

Autosomal dominant polycystic kidney disease (ADPKD), primarily driven by PKD1 or PKD2 mutations, is a prevalent hereditary nephropathy for which large-animal models capturing human renal anatomy and disease tempo remain urgently needed. This study aimed to establish a site-specific human PKD2 (hPKD2) transgenic porcine model as a versatile platform capable of supporting two temporally distinct applications: longitudinal evaluation of whether sustained PKD2 overexpression is sufficient to induce renal pathology, and—should overexpression prove phenotypically silent—future crossbreeding-based functional rescue of PKD2-knockout lines.

Methods and Results

A CRISPR/Cas9-mediated, homology-recombination-independent strategy was employed to target full-length hPKD2 cDNA into the porcine pH11 safe harbor locus. Somatic cell nuclear transfer yielded five F0 transgenic founders, and natural mating of two founders with wild-type sows produced four F1 transgenic offspring, confirming stable germline transmission. Quantitative real-time PCR and whole-genome sequencing validated single-copy, site-specific transgene integration at the designated locus. Robust hPKD2 mRNA and FLAG-tagged polycystin-2 (PC-2) expression were detected in renal and other tissues across both generations. During the initial 12-month monitoring period, serum blood urea nitrogen and creatinine levels remained within normal ranges and no gross histological abnormalities were evident. However, given that these biomarkers are insensitive to early-stage renal impairment, extended observation with more comprehensive phenotyping is required before definitive conclusions regarding renal function can be drawn.

Conclusions

A transgenic porcine model with stable, single-copy hPKD2 integration at the pH11 safe harbor locus was successfully generated and shown to permit germline transmission. This platform provides a foundation for long-term investigation of PKD2 overexpression pathophysiology and, alternatively, for functional complementation of PKD2-deficient models, thereby advancing both mechanistic and translational ADPKD research.