Produção de embriões transgênicos bovinos por meio de microinjeção de vetores lentivirais ou transferência nuclear de células somáticas

• Ano de defesa: 2012
• Autor(a) principal: Arroyo, Rafael José Otero
• Orientador(a): Costa, Eduardo Paulino da
• Banca de defesa: Iguma, Lilian Tamy , Veloso, Cristina Mattos
• Tipo de documento: Tese
• Tipo de acesso: Acesso aberto
• Idioma: por
• Instituição de defesa: Universidade Federal de Viçosa
• Programa de Pós-Graduação: Doutorado em Medicina Veterinária
• Departamento: Biotecnologia, diagnóstico e controle de doenças; Epidemiologia e controle de qualidade de prod. de
• País: BR
• Palavras-chave em Português: Embriões bovinos; Transgenia; Clonagem
• Palavras-chave em Inglês: Bovine embryos; Transgenesis; Cloning
• Área do conhecimento CNPq: CNPQ::CIENCIAS AGRARIAS::MEDICINA VETERINARIA::REPRODUCAO ANIMAL
• Link de acesso: http://locus.ufv.br/handle/123456789/1453

In these days, the main barriers for production of transgenic animals are the identification of efficient systems of transgenic, such as different mechanisms on finding gene expression. Based on the idea of searching for different ways of developing transgenic animals, the objective of this research was to produce transgenic bovine embryos using the lentiviral vector of microinjection or nuclear transfers of genetically modified somatic cells, which have the finality of developing and quickly selecting animals from transgenic bovine embryos. This research was conducted in two different stages. Stage I consisted on establishing and producing a lentiviral vector which codified GFP protein. Stage II consisted on conducting three different experiments. On experiment I, a microinjection was developed on the lentiviral vector in the perivitelline space of the bovine ovocites before fecundation (T4), obtaining 5,26 percent of blastocites. When compared with the controle (T1) (complex ovoocites with cumulus-ovocites and without manipulation) the percetage was lower (P< 0,05), control without microinjection (T2), or a control with microinjections and Talp medium (T3). On all the microinjected embryos with the lentiviral vectors, a gene expression of GFP (Green Fluourocent Protein) was detected. On experiment II, a microinjection of the lentiveral vector on the perivitelline space of the of the bovine zygote was evaluated six hours after fecundation. The microinjected zygotes with the lentiveral vector (T4), on in vitro cultivars, blastocite rates day seven and day eight, 3,4% and 3,8% respectively. This values were below (P<0,05) in comparison with the control (T1) (complex ovoocites with cumulus-ovocites and without manipulation), controle without microinjection (T2), or a control with microinjections and Talp medium (T3). It was possible to recognize the microinjected zygotes with the GFP gene on blastocites on day seven (75%) and day eight (77,7%). On experiment III, the triconstatitina A (TSA) was evaluated producing the transgenic bovine embryos using a nuclear transfer with genetically modified somatic cells with lentiveral vector. There was no difference (P> 0.05) in blastocyst rate of embryos treated with trichostatin (T1) (18.1%) when compared with untreated embryos (T2) (6.8%). When the rate was calculated from cleaved zygotes, production was higher (P <0.05) for embryos treated with trichostatin. In both treatments, all generated blastocysts expressed the GFP gene. In this research the microinjection with the lentiviral vector on ovocites and zygotes after six hours later pos-fecundation affected the rates of embryo development. It was proved that the microinjection using the lentiviral vectors on ovocites and zygotes cause a higher expression of green fluorescent protein (GFP). Additionally, the TSA contributed with a better development of cloned embryos on genetically modified cells.