title: NanoSMGT: transgene transmission into bovine embryos using halloysite clay nanotubes or nanopolymer to improve transfection efficiency.
abstract: The objectives were to investigate whether: 1) nanotransfectants are more effective than other common transfection methods for SMGT; 2) NanoSMGT is able to transmit exogenous DNA molecules to bovine embryos; and 3) halloysite clay nanotubes (HCNs) can be used as a transfection reagent to improve transgene transmission. Four transfection systems were used: naked DNA (without transfectant), lipofection, nanopolymer, and halloysite clay nanotubes. Plasmid uptake by sperm and its transfer to embryos were quantified by conventional and real-time PCR, as well as EGFP expression by fluorescence microscopy. Furthermore, sperm motility and viability, and embryo development were investigated. Mean number of plasmids taken up was affected (P < 0.05) by transfection procedure, with the nanopolymer being the most effective transfectant (∼ 153 plasmids per spermatozoon). None of the treatments affected sperm motility or viability. The mean number of plasmids transmitted to four-cell stage embryos was higher (P < 0.05) in nanopolymer and HCNs than liposomes and naked DNA groups. The number of embryos carrying the transgene increased from 8-10% using naked DNA or liposomes to 40-45% using nanopolymer or HCN as transfectants (P < 0.05). There were no significant differences among transfection procedures regarding blastocyst formation rate of resulting embryos. However, no EGFP-expressing embryo was identified in any treatment. Therefore, nanotransfectants improved transgene transmission in bovine embryos without deleterious effects on embryo development. To our knowledge, this was the first time that bovine embryos carrying a transgene were produced by NanoSMGT.
authors.affiliation: Laboratório de Embriologia Molecular e Transgênese, Núcleo de Biotecnologia, Centro de Desenvolvimento Tecnológico, Universidade Federal de Pelotas, Pelotas, RS, Brazil.
authors.forename: Vinicius Farias
authors.initials: VF
authors.lastname: Campos
authors.forename: Priscila Marques Moura
authors.initials: PM
authors.lastname: de Leon
authors.forename: Eliza Rossi
authors.initials: ER
authors.lastname: Komninou
authors.forename: Odir Antônio
authors.initials: OA
authors.lastname: Dellagostin
authors.forename: João Carlos
authors.initials: JC
authors.lastname: Deschamps
authors.forename: Fabiana Kömmling
authors.initials: FK
authors.lastname: Seixas
authors.forename: Tiago
authors.initials: T
authors.lastname: Collares
mesh_terms: D000538:Aluminum Silicates / Q000737:chemistry*; D000818:Animals; D030801:Animals, Genetically Modified; D002417:Cattle / Q000196:embryology*; D000077215:Clay; D005260:Female; D005307:Fertilization in Vitro; D008081:Liposomes; D049329:Nanostructures; D014162:Transfection / Q000379:methods / Q000662:veterinary*
chemical_list: D000538:Aluminum Silicates; D008081:Liposomes; D000077215:Clay