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<p>Long-term preservation of genetic lines is crucial for animal research. Current methods heavily rely on cryopreservation of haploid sperm, whereas diploid germline stem cells (GSCs) may provide a superior alternative. Here, using zebrafish, we establish an integrated platform combining in vivo expansion of GSCs (eGSCs), cryopreservation and direct genetic recovery. The eGSCs are amplified in <i>cyp11a2</i><sup><i>-/-</i></sup> mutants and exhibit a total number of about 300-fold higher than conventional GSCs after cryopreservation and thawing. The eGSCs achieve a success rate of 82% after transplantation, compared with less than 1% for conventional GSCs. Furthermore, we develop the GSC-deficient <i>nanos2</i><sup><i>-/-</i></sup> mutant as an ideal host for eGSC transplantation, enabling efficient production of both sperm and oocytes within a single maturation period. This allows rapid recovery of maternal-zygotic mutants directly in the F1 generation. Overall, our strategy provides a robust platform for preserving and restoring zebrafish homozygous mutants, opening new opportunities for zebrafish research.</p>

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Efficient recovery of zebrafish maternal-zygotic mutants from in vivo expanded and cryopreserved germline stem cells

  • 期刊论文

  • 出版模式：

    开放获取

  • 发表日期：

    2026年6月15日

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  • Yaqing Wang,
  • Rui Qu,
  • Xiaosi Wang,
  • Yongkang Hao,
  • Jiajie Deng,
  • Houpeng Wang,
  • Yonghua Sun

摘要

Long-term preservation of genetic lines is crucial for animal research. Current methods heavily rely on cryopreservation of haploid sperm, whereas diploid germline stem cells (GSCs) may provide a superior alternative. Here, using zebrafish, we establish an integrated platform combining in vivo expansion of GSCs (eGSCs), cryopreservation and direct genetic recovery. The eGSCs are amplified in cyp11a2-/- mutants and exhibit a total number of about 300-fold higher than conventional GSCs after cryopreservation and thawing. The eGSCs achieve a success rate of 82% after transplantation, compared with less than 1% for conventional GSCs. Furthermore, we develop the GSC-deficient nanos2-/- mutant as an ideal host for eGSC transplantation, enabling efficient production of both sperm and oocytes within a single maturation period. This allows rapid recovery of maternal-zygotic mutants directly in the F1 generation. Overall, our strategy provides a robust platform for preserving and restoring zebrafish homozygous mutants, opening new opportunities for zebrafish research.