Efficient selection of transgenic mouse embryos using EGFP as a marker gene

Area of Science:

• Molecular Biology
• Developmental Biology
• Genetics

Background:

• Efficiently identifying transgenic embryos is critical for genetic research and animal model development.
• Traditional methods for transgenic identification can be time-consuming and may impact embryo viability.

Purpose of the Study:

• To establish and validate a reliable method for selecting transgenic preimplantation embryos using Enhanced Green Fluorescent Protein (EGFP) as a marker.
• To assess the impact of EGFP expression monitoring on embryo developmental potential.

Main Methods:

• Pronuclear microinjection of the CMV/beta-actin/EGFP fusion gene into mouse embryos.
• In vitro culture of embryos to morula or blastocyst stages.
• Fluorescence microscopy for EGFP expression observation.
• Modified PCR analysis (Dpn I and Bal 31 digestion) for transgenic confirmation.

Main Results:

• EGFP expression was readily detectable via fluorescence microscopy.
• No significant damage to embryo developmental ability was observed after 30 min of EGFP excitation.
• Embryos with uniform EGFP expression were confirmed as transgenic, while mosaic expression often indicated non-transgenic status.
• Transgenic offspring rates were 77% for uniformly EGFP-expressing embryos versus 21.4% for mosaic ones.

Conclusions:

• EGFP is a highly useful and reliable marker for selecting transgenic embryos.
• Confirming uniform EGFP expression across all cells is essential for accurate identification of truly transgenic animals.