Generating chimeric mice from embryonic stem cells via vial coculturing or hypertonic microinjection.

Area of Science:

• Reproductive Biology
• Developmental Biology
• Stem Cell Biology

Background:

• Generating fertile chimeric mice from embryonic stem cells (ESCs) is crucial for validating pluripotency.
• Conventional methods like aggregation and microinjection have limitations, including technical complexity and systemic constraints.

Purpose of the Study:

• To introduce and evaluate two novel, efficient, and accessible methods for producing chimeric mice.
• To provide alternatives to existing techniques for generating ESC-derived F0 mice with high chimerism and germline transmission.

Main Methods:

• Vial coculturing: Co-culturing denuded 2.5-day post-coitum (dpc) embryos with ESCs in vials for approximately 3 hours.
• Hypertonic microinjection: Microinjecting ESCs into the subzonal space of 2.5-dpc embryos using a hypertonic sucrose medium.

Main Results:

• Vial coculturing yields high levels of chimerism (including 100% coat color) and germline transmission, with fewer limitations than traditional methods.
• Hypertonic microinjection efficiently generates viable, healthy, and fertile chimeric mice with 100% coat color chimerism.
• Both methods are effective for both ESCs and induced pluripotent stem cells (iPSCs).

Conclusions:

• Vial coculturing and hypertonic microinjection are reliable and efficient alternatives for producing germline chimeric or F0 mice.
• These novel methods overcome limitations of conventional techniques, offering simplicity, cost-effectiveness, and reproducibility.
• The methods are suitable for mass production of chimeric embryos and applicable to both ESCs and iPSCs.