title: Exposure to DNA is insufficient for in vitro transgenesis of live bovine sperm and embryos.
abstract: Transgenic mammals have been produced using sperm as vectors for exogenous DNA (sperm-mediated gene transfer (SMGT)) in combination with artificial insemination. Our study evaluated whether SMGT could also be achieved in combination with IVF to efficiently produce transgenic bovine embryos. We assessed binding and uptake of fluorescently labelled plasmids into sperm in the presence of different concentrations of dimethyl sulphoxide or lipofectamine. Live motile sperm displayed a characteristic punctuate fluorescence pattern across their entire surface, while uniform postacrosomal fluorescence was only apparent in dead sperm. Association with sperm or lipofection reagent protected exogenous DNA from DNase I digestion. Following IVF, presence and expression of episomal and non-episomal green fluorescent protein (GFP)-reporter plasmids was monitored in oocytes and embryos. We found no evidence of intracellular plasmid uptake and none of the resulting zygotes (n=96) and blastocysts were GFP positive by fluorescence microscopy or genomic PCR (n=751). When individual zona-free oocytes were matured, fertilised and continuously cultured in the presence of episomal reporter plasmids until the blastocyst stage, most embryos (38/68=56%) were associated with the exogenous DNA. Using anti-GFP immunocytochemistry (n=48) or GFP fluorescence (n=94), no GFP expression was detected in blastocysts. By contrast, ICSI resulted in 18% of embryos expressing the GFP reporter. In summary, exposure to DNA was an inefficient technique to produce transgenic bovine sperm or blastocysts in vitro.
authors.affiliation: Young Researchers Club, Khorasgan Branch, Islamic Azad University, Isfahan, Iran.
authors.forename: Shahin
authors.initials: S
authors.lastname: Eghbalsaied
authors.forename: Kamran
authors.initials: K
authors.lastname: Ghaedi
authors.forename: Götz
authors.initials: G
authors.lastname: Laible
authors.forename: Sayed Morteza
authors.initials: SM
authors.lastname: Hosseini
authors.forename: Mohsen
authors.initials: M
authors.lastname: Forouzanfar
authors.forename: Mehdi
authors.initials: M
authors.lastname: Hajian
authors.forename: Fleur
authors.initials: F
authors.lastname: Oback
authors.forename: Mohammad H
authors.initials: MH
authors.lastname: Nasr-Esfahani
authors.forename: Björn
authors.initials: B
authors.lastname: Oback
mesh_terms: D000818:Animals; D030801:Animals, Genetically Modified / Q000235:genetics*; D002417:Cattle / Q000235:genetics*; D002478:Cells, Cultured; D004247:DNA / Q000378:metabolism / Q000494:pharmacology*; D004622:Embryo, Mammalian / Q000187:drug effects* / Q000378:metabolism; D005260:Female; D005307:Fertilization in Vitro / Q000379:methods; D018014:Gene Transfer Techniques; D049452:Green Fluorescent Proteins / Q000378:metabolism; D066298:In Vitro Techniques; D007315:Insemination, Artificial / Q000379:methods; D008297:Male; D010957:Plasmids; D020554:Sperm Injections, Intracytoplasmic / Q000379:methods; D013094:Spermatozoa / Q000187:drug effects* / Q000378:metabolism; D014162:Transfection
chemical_list: D049452:Green Fluorescent Proteins; D004247:DNA