Modifications to improve the efficiency of zona-free mouse nuclear transfer

Area of Science:

• Reproductive Biology
• Developmental Biology
• Animal Biotechnology

Background:

• Nuclear transfer is a key technique in reproductive biology.
• Improving the efficiency of nuclear transfer is crucial for its wider application.
• Previous methods faced challenges with embryo survival and fusion rates.

Purpose of the Study:

• To enhance the efficiency of the zona-free nuclear transfer technique in mice.
• To identify specific modifications that improve embryo survival and development post-transfer.
• To optimize conditions for successful reconstruction using embryonic stem cells.

Main Methods:

• Modified the timing between cumulus removal and zona pellucida digestion.
• Utilized acid Tyrode's solution for zona pellucida removal instead of pronase.
• Performed electrofusion at room temperature (21°C).
• Cultured embryos in groups (12-15) using a "well of the wells" system.

Main Results:

• Acid Tyrode's allowed embryo survival during activation without calcium, unlike pronase.
• Group culture of parthenogenetic embryos increased development to blastocyst compared to individual culture.
• Achieved a 1.5-fold increase in fusion rate and development to morula/blastocyst from reconstructed embryos.
• Attained a 2.4-fold increase in overall efficiency from oocyte to morula/blastocyst stages.

Conclusions:

• The modified zona-free nuclear transfer protocol significantly increases efficiency.
• Specific procedural adjustments, including zona digestion method and culture conditions, are critical for success.
• The optimized technique holds promise for improved applications in assisted reproduction and developmental studies.