Liposome-mediated uptake of exogenous DNA by equine spermatozoa and applications in sperm-mediated gene transfer.

The research discusses a method using equine spermatozoa to introduce exogenous DNA into early horse embryos, a potential technique for creating transgenic horses. Despite finding traces of the introduced DNA in developing embryos, there was no evidence of its expression.

Research Objective and Methodology

• The aim of this research was to assess whether exogenous DNA, specifically, the enhanced green fluorescent protein (pEGFP), could be taken up by horse sperm and if such sperm could successfully introduce this DNA into early horse embryos.
• This was done by washing semen and incubating it with 32P-pEGFP, either with or without lipofection, a process that enables more efficient uptake of the DNA.
• Sperm was then transfected with fluorescently-labeled DNA (Alexa647-pEGFP) and alterations in sperm viability and DNA uptake were studied.
• The researchers then inseminated mares with the pEGFP-transfected sperm and recovered resulting embryos.
• The embryonic expression of pEGFP was scrutinized using epifluorescence microscopy, while the presence of pEGFP DNA and mRNA was checked using PCR and RT-PCR respectively.

Results

• The results showed that liposome-mediated transfection helped significantly in the incorporation of 32P-pEGFP into the sperm, as compared to controls.
• Flow cytometric assessment of spermatozoa after transfusion with Alexa647-pEGFP showed that there was a linear increase in the proportion of live, Alexa647+ spermatozoa with an increase in DNA concentrations.
• On inseminating with the transfected spermatozoa, eight embryos were successfully recovered.
• Although the EGFP gene was found in two out of five analyzed embryos, there was no concrete evidence of EGFP expression in the embryos.

Conclusions

• The research concluded that liposome-mediated transfection greatly improved the uptake of exogenous DNA by horse spermatozoa and didn’t impact the sperm’s viability, acrosomal integrity or fertility.
• While traces of the EGFP transgene were detected in a number of the embryos, no evidence was found to show that the EGFP was being expressed in them.
• Thus, the potential of using sperm-mediated gene transfer as a possible technique for creating transgenic equids is suggested but not yet confirmed.