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

## TL;DR

This study presents a safe and effective method for delivering genes to the testes of newborn mice using isoflurane anesthesia and simple lab equipment.

## Contribution

A novel, safe isoflurane-based anesthesia protocol for intra-testicular gene delivery in neonatal mice is introduced.

## Key findings

- The isoflurane-based method achieved over 90% postoperative survival in neonatal mice.
- Gene delivery efficiently targeted interstitial Leydig cells but showed limited transfection in seminiferous tubules.
- The method enables gene engineering in both somatic and germ cells of neonatal mice.

## Abstract

Newborn mice (up to 6 d after birth) are suitable for genetic manipulations, such as facial vein-mediated injection, owing to their hairless and thin skin. Their small body volumes also facilitate the rapid dissemination of injected solutions, supporting gene engineering-related experiments. However, anesthesia in newborns is challenging because of the potential risks associated with anesthetic agents. Isoflurane inhalation anesthesia is an option, although its effects on brain development remain under investigation. In this study, we established a reproducible protocol for delivering nucleic acids to juvenile mouse testes using a simple isoflurane-based anesthetic system prepared from common laboratory equipment. Using this system, nucleic acids were successfully delivered to juvenile mouse testes via intra-testicular injection, followed by in vivo electroporation. The present isoflurane-based method achieved >90% postoperative survival with normal maternal nursing observations. Gene delivery resulted in limited transfection of seminiferous tubules but efficient interstitial Leydig cell transfection. Thus, gene engineering in somatic and germ cells in neonatal mice will be facilitated using the anesthetic protocol established in this study.