A method for tracing exogenous DNA uptake in live spermatozoa and embryos

Area of Science:

• Reproductive Biology
• Molecular Biology
• Biotechnology

Background:

• Sperm-mediated gene transfer (SMGT) is a promising technique for creating transgenic animals.
• Current SMGT methods suffer from low efficiency due to inconsistent sperm DNA binding and uptake.
• There is a need for reliable methods to assess sperm's ability to bind and internalize exogenous DNA.

Purpose of the Study:

• To develop and validate a novel method for evaluating sperm's capacity to bind exogenous DNA post-incubation.
• To utilize Cy5-labeled DNA for real-time tracing of exogenous DNA within sperm and zygotes.
• To optimize DNA concentrations for enhanced sperm DNA uptake in SMGT.

Main Methods:

• Co-incubation of spermatozoa with Cy5-labeled DNA at various concentrations (0, 1, 5, 10, 100 μg/mL).
• Assessment of exogenous DNA binding and uptake by spermatozoa using fluorescence microscopy after 30 minutes incubation at 37°C.
• Detection of DNA fluorescence signal in resulting zygotes post-fertilization.

Main Results:

• Spermatozoa exhibited higher binding and uptake of Cy5-DNA at concentrations of 10 μg/mL and 100 μg/mL compared to lower concentrations.
• Fluorescence signals from Cy5-DNA were successfully detected in zygotes derived from sperm incubated with 10 μg/mL and 100 μg/mL DNA.
• The Cy5-labeling method provided a simple, convenient, and real-time observation of exogenous DNA transfer.

Conclusions:

• The developed method using Cy5-labeled DNA is effective for evaluating sperm's exogenous DNA binding and uptake capacity.
• Optimized DNA concentrations (10 and 100 μg/mL) significantly improve DNA transfer efficiency in SMGT.
• This technique offers a valuable tool for advancing SMGT efficiency and understanding DNA transfer mechanisms in reproductive biotechnology.