Rescue of germline transmission from chimeras by IVF after sperm analysis

Area of Science:

• Genetics
• Reproductive Biology
• Animal Models

Background:

• Germline transmission (GLT) is crucial for creating genetically modified mouse lines.
• When natural mating fails, sperm harvesting from chimeras for assisted reproductive technologies like in vitro fertilization (IVF) is an option.
• A rational approach to select chimeras for these efforts is lacking.

Purpose of the Study:

• To assess sperm production, quality, and genotype from male chimeras that failed GLT via natural mating.
• To establish criteria for selecting chimeras for IVF to rescue GLT.
• To correlate coat color chimerism with sperm genotype and assess IVF success rates.

Main Methods:

• Male chimeras were generated using microinjection of gene-targeted embryonic stem cells.
• Epididymal sperm were harvested and analyzed for production, quality, and LacZ genotype.
• Coat color chimerism was recorded, and IVF was performed using selected sperm.

Main Results:

• 80% of chimeras failed GLT by natural mating; 63% of these had no sperm, 15% had poor quality sperm.
• A strong positive linear correlation (R² = 0.95) was found between coat color chimerism and LacZ-positive sperm percentage.
• IVF successfully rescued GLT for 19 out of 56 genes using sperm from selected chimeras.

Conclusions:

• Most chimeras failing natural GLT are infertile due to failed spermatogenesis.
• Coat color chimerism alone is insufficient for selecting chimeras for IVF.
• Combined assessment of coat color, sperm quality, and genotype optimizes chimera selection for rescuing GLT via IVF.