Project Objectives

• Develop genetically sterile host embryos to enable exclusive pluripotent stem cell (PSC)‑derived germline transmission.
• Use a Y‑linked fluorescent reporter to determine embryo sex at the two‑cell stage and ensure PSC–embryo sex matching.
• Reduce the number of surplus animals typically generated during breeding and genotyping.
• Replace surgical vasectomy with genetically sterile males for creating foster females.
• Generate proof‑of‑concept strains.

3Rs Impact

• Significantly reduces the number of surplus animals by enabling exclusive PSC germline contribution and eliminating non‑transgenic offspring.
• Refines animal procedures by reducing or eliminating biopsy‑based genotyping across generations.
• Replaces surgically vasectomized males with genetically sterile lines, removing an invasive procedure associated with pain and distress.
• Improves efficiency of transgenic production, reducing breeding rounds and overall animal use.
• Prevents production of sex‑mismatched or infertile chimeras through early embryo sex selection.

Background

Generating transgenic animals is essential for modelling disease, studying gene function, and evaluating therapies, but current workflows are slow, inefficient, and produce large numbers of surplus animals. Standard procedures require injecting pluripotent stem cells (PSCs) or CRISPR reagents into early-stage embryos, followed by extensive breeding and genotyping to identify animals that successfully carry the desired transgene.

Many animals born in this process do not contribute to the germline and must be culled, while others undergo invasive biopsies for PCR‑based verification. Female chimeras and sex‑mismatched chimeras are often unusable, further increasing animal waste. A major additional burden comes from the use of surgically vasectomized males, which are required to generate foster females for embryo transfer. Vasectomy is painful, carries post‑operative risks, and must be repeated routinely.

This project proposes a transformative alternative: using genetically sterile mouse strains that produce embryos lacking competing germ cells, enabling exclusive PSC‑derived germline transmission. A Y‑chromosome fluorescent reporter allows early embryo sexing, enabling sex‑matching between PSCs and host embryos. Together, these innovations streamline the pipeline, reduce breeding, eliminate non‑productive chimeras, and replace surgical vasectomy with a genetic solution.