A simple and efficient method to transfect small interference RNA into bovine SCNT embryos

Area of Science:

• Reproductive Biology
• Molecular Biology
• Genetics

Background:

• RNA interference (RNAi) is crucial for gene function analysis.
• Conventional methods like microinjection and electroporation are used for molecule delivery into oocytes/embryos.
• Efficient siRNA delivery into bovine SCNT embryos is challenging.

Purpose of the Study:

• To develop a simple and effective method for transfecting siRNA into bovine SCNT embryos.
• To combine siRNA transfection with electrofusion during SCNT.
• To assess the efficiency and impact of this combined method on SCNT embryos.

Main Methods:

• A novel approach combining siRNA transfection and electrofusion for SCNT embryos.
• Utilizing platinum microelectrodes for electrofusion and simultaneous siRNA delivery.
• Employing CY3-labeled siRNA targeting DNA methyltransferase-1 (DNMT1) to confirm transfection efficacy.

Main Results:

• Successful transfection of siRNA into bovine SCNT embryos was confirmed by CY3 fluorescence.
• Significant downregulation of DNMT1 mRNA and protein levels was observed.
• Global DNA methylation levels were reduced in SCNT embryos post-transfection.
• No significant adverse effects on embryo survival or developmental rates were detected.

Conclusions:

• The developed method effectively transfects siRNA into bovine SCNT embryos, allowing for functional gene studies.
• This combined approach does not compromise the survival or developmental potential of preimplantation SCNT embryos.
• This technique offers a promising tool for genetic manipulation in bovine SCNT embryos.