Optimizing injection time of GFP plasmid into sheep zygote

Area of Science:

• Animal Biotechnology
• Reproductive Biology
• Genetics

Background:

• Generating transgenic animals is vital for research and agriculture.
• Microinjection into oocytes or zygotes is a key technique for creating transgenic animals.
• Specific protocols for sheep microinjection, particularly timing, require further optimization.

Purpose of the Study:

• To determine the optimal time point for cytoplasmic microinjection of exogenous DNA in Sarda sheep.
• To evaluate the efficiency of generating transgenic sheep using different microinjection time points.

Main Methods:

• Sarda sheep oocytes were matured in vitro (IVM) and fertilized in vitro (IVF).
• A Green Fluorescent Protein (GFP) plasmid was microinjected into MII oocytes and zygotes at various post-insemination intervals (6-14 hpi).
• Cleavage rates, blastocyst rates, and GFP expression were assessed in the resulting embryos.

Main Results:

• No significant difference in cleavage rates was observed across groups.
• Blastocyst rates were significantly lower in zygotes injected at all time points compared to MII oocytes and controls.
• The highest proportion of GFP-positive embryos (4%) was observed following injection at 8-10 hpi, with some mosaic embryos reaching the blastocyst stage.

Conclusions:

• Cytoplasmic microinjection of GFP plasmid at 8-10 hours post-insemination is an optimized time point for producing transgenic sheep.
• This optimized timing facilitates higher efficiency in generating genetically modified sheep embryos.
• Further studies can build upon this optimized protocol for advanced transgenic sheep applications.