title: Techniques for augmentation of exogenous DNA uptake by ovine spermatozoa.
abstract: Sperm mediated gene transfer can be an inexpensive and simple method in animal transgenesis; however its efficiency is poor, mainly due to the spermatozoa's lesser uptake of exogenous DNA. In the present study, the effects of lipofection and other augmentation techniques, such as sperm freezing and spermatozoa treatment with triton X100 and DMSO, on exogenous DNA uptake by sheep spermatozoa and motility of sperms with plasmid uptake were evaluated. In the first experiment, ram sperms were incubated with a complex of rhodamine labeled plasmid (p-EGFP) and Lipofectamine 2000(TM). In the second, spermatozoa were treated with Triton X-100(TM) or DMSO or were frozen without cryoprotectant. The results indicated that there was no significant difference (P<0.05) in the transfection rates and in the uptake intensity of lipofected sperms with 300 and 600 ng of plasmid in comparison with control group, i.e. transfected without lipofectamine. Furthermore, lipofection could not improve sperm motility during true plasmid uptake. Almost all of triton X100 treated and frozen-thawed spermatozoa had absorbed foreign DNA, though all were immotile. In spermatozoa treated with 0.1% DMSO, plasmid absorption rate (69.40%) was significantly higher (P<0.05) than untreated spermatozoa (57.80%), but sperm motility was not significantly different from control group. In conclusion, lipofectamine(®) 2000 could neither improve transfection rate, nor support motility in transfected sperms. The methods inducing membrane disruption like, freeze-thaw and triton X100 treatment, can be used in ICSI-sperm mediated gene transfer without the need for sperm selection, provided that they cause no damage to sperm nucleus.
authors.affiliation: Department of Clinical Sciences, Faculty of Veterinary Medicine, University of Tehran, Tehran, Iran;
authors.forename: K
authors.initials: K
authors.lastname: Hoseini Pajooh
authors.affiliation: Department of Clinical Sciences, Faculty of Veterinary Medicine, University of Tehran, Tehran, Iran;
authors.forename: P
authors.initials: P
authors.lastname: Tajik
authors.affiliation: Molecular Medicine Department, Biotechnology Research Center, Pasteur Institute of Iran, Tehran, Iran.
authors.forename: M
authors.initials: M
authors.lastname: Karimipoor
authors.affiliation: Molecular Medicine Department, Biotechnology Research Center, Pasteur Institute of Iran, Tehran, Iran.
authors.forename: M
authors.initials: M
authors.lastname: Behdani
keywords: Lipofection; SMGT; Sheep; Sperm; Transfection