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A Simple and Safe Protocol for Intra-Testicular Gene Delivery in Neonatal Mice Using a Convenient Isoflurane-Based

Kazunori Morohoshi1, Miho Ohba1, Masahiro Sato2

  • 1Division of Biomedical Engineering, National Defense Medical College Research Institute, 3-2 Namiki, Tokorozawa, Saitama 359-8513, Japan.

Biotech (Basel (Switzerland))
|November 24, 2025
PubMed
Summary

This study presents a safe and effective isoflurane anesthesia protocol for gene delivery in newborn mouse testes. The method enables efficient gene engineering in somatic and germ cells, facilitating future research.

Keywords:
anesthetic devicegenome-engineeringinhalation anesthesia systemintra-testicular gene deliveryisofluranenewborntestis

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Area of Science:

  • Neonatal mouse models
  • Gene delivery techniques
  • Anesthesiology in research animals

Background:

  • Newborn mice are ideal for genetic manipulation due to their physical characteristics.
  • Anesthesia in neonatal research subjects poses significant risks.
  • Isoflurane anesthesia's impact on neonatal brain development requires further investigation.

Purpose of the Study:

  • To establish a reproducible anesthetic protocol for nucleic acid delivery to juvenile mouse testes.
  • To evaluate the safety and efficacy of an isoflurane-based anesthetic system for neonatal gene engineering.
  • To assess gene transfection efficiency in different testicular cell types.

Main Methods:

  • Development of a simple isoflurane-based anesthetic system using standard laboratory equipment.
  • Intra-testicular injection of nucleic acids followed by in vivo electroporation in juvenile mice.
  • Assessment of postoperative survival rates and maternal nursing behavior.

Main Results:

  • The isoflurane-based anesthetic protocol achieved over 90% postoperative survival.
  • Gene delivery resulted in efficient transfection of interstitial Leydig cells.
  • Limited transfection was observed in seminiferous tubules.

Conclusions:

  • The established isoflurane anesthesia protocol is safe and reproducible for neonatal mouse testicular gene delivery.
  • This method facilitates gene engineering in both somatic (Leydig) and germ cells in neonatal mice.
  • The protocol supports advancements in genetic manipulation research in young animal models.