title: Effect of transfection and co-incubation of bovine sperm with exogenous DNA on sperm quality and functional parameters for its use in sperm-mediated gene transfer.
abstract: Sperm-mediated gene transfer (SMGT) is based on the capacity of sperm to bind exogenous DNA and transfer it into the oocyte during fertilization. In bovines, the progress of this technology has been slow due to the poor reproducibility and efficiency of the production of transgenic embryos. The aim of the present study was to evaluate the effects of different sperm transfection systems on the quality and functional parameters of sperm. Additionally, the ability of sperm to bind and incorporate exogenous DNA was assessed. These analyses were carried out by flow cytometry and confocal fluorescence microscopy, and motility parameters were also evaluated by computer-assisted sperm analysis (CASA). Transfection was carried out using complexes of plasmid DNA with Lipofectamine, SuperFect and TurboFect for 0.5, 1, 2 or 4 h. The results showed that all of the transfection treatments promoted sperm binding and incorporation of exogenous DNA, similar to sperm incorporation of DNA alone, without affecting the viability. Nevertheless, the treatments and incubation times significantly affected the motility parameters, although no effect on the integrity of DNA or the levels of reactive oxygen species (ROS) was observed. Additionally, we observed that transfection using SuperFect and TurboFect negatively affected the acrosome integrity, and TurboFect affected the mitochondrial membrane potential of sperm. In conclusion, we demonstrated binding and incorporation of exogenous DNA by sperm after transfection and confirmed the capacity of sperm to spontaneously incorporate exogenous DNA. These findings will allow the establishment of the most appropriate method [intracytoplasmic sperm injection (ICSI) or in vitro fertilization (IVF)] of generating transgenic embryos via SMGT based on the fertilization capacity of transfected sperm.
authors.affiliation: Laboratory of Reproduction,Centre of Reproductive Biotechnology (CEBIOR-BIOREN),Universidad de La Frontera,Temuco,Chile.
authors.forename: María Elena
authors.initials: ME
authors.lastname: Arias
authors.affiliation: Laboratory of Reproduction,Centre of Reproductive Biotechnology (CEBIOR-BIOREN),Universidad de La Frontera,Temuco,Chile.
authors.forename: Esther
authors.initials: E
authors.lastname: Sánchez-Villalba
authors.affiliation: Laboratory of Reproduction,Centre of Reproductive Biotechnology (CEBIOR-BIOREN),Universidad de La Frontera,Temuco,Chile.
authors.forename: Andrea
authors.initials: A
authors.lastname: Delgado
authors.affiliation: Laboratory of Reproduction,Centre of Reproductive Biotechnology (CEBIOR-BIOREN);Department of Agricultural Production; and Department of Agricultural Sciences and Natural Resources,Faculty of Agriculture and Forestry Sciences,Universidad de La Frontera,Montevideo 0870,P.O. Box 54-D,Temuco,Chile.
authors.forename: Ricardo
authors.initials: R
authors.lastname: Felmer
mesh_terms: D000177:Acrosome; D000818:Animals; D030801:Animals, Genetically Modified; D002417:Cattle; D004247:DNA / Q000008:administration & dosage / Q000378:metabolism*; D005307:Fertilization in Vitro / Q000379:methods; D018014:Gene Transfer Techniques; D008055:Lipids; D008297:Male; D053078:Membrane Potential, Mitochondrial; D010957:Plasmids / Q000235:genetics; D017382:Reactive Oxygen Species / Q000378:metabolism; D013081:Sperm Motility; D013094:Spermatozoa / Q000502:physiology*; D014162:Transfection / Q000379:methods*
chemical_list: D008055:Lipids; C086724:Lipofectamine; D017382:Reactive Oxygen Species; D004247:DNA
keywords: Cattle; Embryos; Spermatozoa; Transfection; Transgenic