Liposome-mediated uptake of exogenous DNA by equine spermatozoa and applications in sperm-mediated gene transfer.
BACKGROUND
Sperm-mediated gene transfer has been reported as a method for production of transgenic animals in a variety of species, and this technique represents a possible method for production of transgenic equids.


OBJECTIVE
To evaluate the uptake of exogenous DNA (enhanced green fluorescent protein; pEGFP) by equine spermatozoa and to assess the ability of transfected spermatozoa to introduce this transgene into early equine embryos.


METHODS
To evaluate incorporation of pEGFP into equine spermatozoa, washed spermatozoa were incubated with 32P-pEGFP, with or without lipofection. Spermatozoa were also transfected with fluorescently-labelled DNA (Alexa647-pEGFP) and changes in sperm viability and DNA uptake were assessed. Mares were inseminated with pEGFP-transfected spermatozoa and embryos recovered. Expression of pEGFP was assessed by epifluorescence microscopy of embryos, and the presence of pEGFP DNA and mRNA was assessed by PCR and RT-PCR, respectively.


RESULTS
Liposome-mediated transfection increased the incorporation of 32P-pEGFP into spermatozoa compared to controls. Flow cytometric evaluation of spermatozoa after transfection with Alexa647-pEGFP revealed a linear increase in the proportion of live, Alexa647+ spermatozoa with increasing DNA concentrations. After insemination with transfected spermatozoa, 8 embryos were recovered. There was no evidence of EGFP expression in the recovered embryos; however, PCR analysis revealed evidence of the pEGFP transgene in 2 of 5 embryos analysed.


CONCLUSIONS
The incorporation of exogenous DNA by equine spermatozoa was enhanced by liposome-mediated transfection and this did not adversely affect sperm viability, acrosomal integrity or fertility. Although the EGFP transgene was detected in a proportion of Day 7-10 embryos, there was no evidence of expression of EGFP in these embryos.


CONCLUSIONS
Sperm-mediated gene transfer offers a potential technique for the generation of transgenic equids.
Ball
B A
BA
Department of Population Health and Reproduction, University of California-Davis, Davis, CA 95616, USA.
Sabeur
K
K
Allen
W R
WR
D020101:Acrosome Reaction; D000818:Animals; D030801:Animals, Genetically Modified; D004247:DNA / Q000235:genetics* / Q000378:metabolism; D005260:Female; D018014:Gene Transfer Techniques / Q000662:veterinary*; D049452:Green Fluorescent Proteins; D006736:Horses / Q000196:embryology / Q000235:genetics*; D008081:Liposomes; D008297:Male; D008856:Microscopy, Fluorescence / Q000662:veterinary; D016133:Polymerase Chain Reaction / Q000662:veterinary; D011247:Pregnancy; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000662:veterinary; D013076:Sperm Count / Q000662:veterinary; D013094:Spermatozoa / Q000502:physiology*; D014162:Transfection / Q000379:methods / Q000662:veterinary*
D008081:Liposomes; D049452:Green Fluorescent Proteins; D004247:DNA