title: Expression of fluorescent reporter protein in equine embryos produced through intracytoplasmic sperm injection mediated gene transfer (ICSI-MGT).
abstract: Sperm mediated gene transfer (SMGT) has been reported to be a powerful tool for producing transgenic livestock with applications in biomedicine and agriculture. To date, two studies have reported the production of transgenic equine embryo with, however, low efficiency in blastocyst production and transgene expression. The aim of the present study was to develop a method which allowed the efficient production of transgene-expressing embryos of the equine species through SMGT. To overcome problems due to in vitro fertilization (IVF) in horses, the ICSI procedure was associated with SMGT. The uptake of exogenous DNA in equine spermatozoa was assessed using a spectrophotometric approach and its internalisation using real time PCR and confocal laser scanning microscopy (CLSM). Embryos obtained from the ICSI-MGT procedure were analysed for the expression of eGFP and then for the transmission of the transgene. Our results suggested that the maximal uptake of exogenous DNA in equine spermatozoa occurs from 30 to 60min of co-incubation. Furthermore, real time PCR analysis suggested that the internalisation of exogenous DNA in the highest quality spermatozoa was slightly greater than in those having the poorest quality parameters. Confocal laser scanning microscopy analysis confirmed that exogenous DNA is internalised by membrane intact spermatozoa. In this study, 22 embryos were produced, 8 of which reached the 8-cell stage or greater. Our data confirmed the transmission of the transgene in 86.3% of the cleaved embryos and the expression of the transgene in 25% of the embryos. These data allowed us to affirm that this method is highly efficient in producing equine embryos which are able to express high levels of the exogenous protein.
authors: Department of Veterinary Medical Sciences - DIMEVET, University of Bologna, Via Tolara di Sopra 50, 40064 Ozzano dell'Emilia, Bologna, Italy. Electronic address: andrea.zaniboni3@unibo.it.
authors: Andrea
authors: A
authors: Zaniboni
authors: Barbara
authors: B
authors: Merlo
authors: Augusta
authors: A
authors: Zannoni
authors: Chiara
authors: C
authors: Bernardini
authors: Marialuisa
authors: M
authors: Lavitrano
authors: Monica
authors: M
authors: Forni
authors: Gaetano
authors: G
authors: Mari
authors: Maria Laura
authors: ML
authors: Bacci
mesh_terms: D000818:Animals; D030801:Animals, Genetically Modified / Q000502:physiology*; D004622:Embryo, Mammalian / Q000502:physiology* / Q000648:ultrastructure; D005260:Female; D018014:Gene Transfer Techniques / Q000662:veterinary*; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000502:physiology; D006736:Horses / Q000235:genetics / Q000502:physiology*; D008297:Male; D018613:Microscopy, Confocal / Q000662:veterinary; D012313:RNA / Q000737:chemistry / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction / Q000662:veterinary; D020554:Sperm Injections, Intracytoplasmic / Q000379:methods / Q000662:veterinary*; D013094:Spermatozoa / Q000502:physiology*
chemical_list: D049452:Green Fluorescent Proteins; D012313:RNA