# Pseudopregnant mice generated from Piwil1 deficiency sterile mice

## TL;DR

This study creates sterile male mice using CRISPR/Cas9 to replace vasectomized mice in transgenic research, reducing animal suffering.

## Contribution

A novel CRISPR-generated Piwil1-deficient mouse model is introduced as an ethical alternative to vasectomized mice.

## Key findings

- Piwil1mt/mt males are sterile, while females remain fertile.
- Using Piwil1mt/mt males in pseudopregnant mouse models yields similar offspring numbers as vasectomized males.
- CRISPR/Cas9 effectively modifies the Miwi gene for assisted reproduction research.

## Abstract

Vasectomized mice play a key role in the production of transgenic mice. However, vasectomy can cause great physical and psychological suffering to mice. Therefore, there is an urgent need to find a suitable replacement for vasectomized mice in the production of transgenic mice. In this study, we generated C57BL/6J mice (Piwil1 D633A-INS99, Piwil1mt/mt) with a 99-base insertion in the Miwi (Piwil1) gene using CRISPR/Cas9 technology and showed that Piwil1mt/+ heterozygous mice were normally fertile and that homozygous Piwil1mt/mt males were sterile and females were fertile. Transplantation of normal fertilized eggs into wild pseudopregnant females following mating with Piwil1mt/mt males produced no Piwil1mt/mt genotype offspring, and the number of offspring did not differ significantly from that of pseudopregnant mice following mating and breeding with ligated males. The CRISPR‒Cas9 system is available for generating Miwi-modified mice, and provides a powerful resource to replace ligated males in assisted reproduction research.