Isolation of transfected fibroblast clones for use in nuclear transfer and transgene detection in cattle embryos
Genet. mol. res. (Online)
; 4(4): 812-821, 2005. tab, ilus
Article
em En
| LILACS
| ID: lil-444840
Biblioteca responsável:
BR1.1
ABSTRACT
Transgenesis in cattle has provided numerous opportunities for livestock production. The development of nuclear transfer (NT) technology has improved the production of transgenic livestock. However, the isolation of pure colonies from a single transfection event remains laborious and can be a constraint in the production of transgenic livestock. We used 96-well cell culture plates to isolate cell lineages obtained from a single fibroblast transfected with the pCi-Neo plasmid. Since single mammalian cells do not grow well in fresh medium, we evaluated the use of conditioned medium. The neomycin phosphotransferase gene was detected in isolated colonies and NT embryos were produced from these cells. Multiplex-PCR assays were performed to detect the transfected fragment as well as autosomal satellite DNA in single NT embryos. This approach provided a reliable method for isolating transfected mammalian cells and for diagnosing the incorporation of desirable vectors in NT embryos. This method can reduce the time and cost of transgenic livestock production.
Palavras-chave
Texto completo:
1
Coleções:
01-internacional
Base de dados:
LILACS
Assunto principal:
Bovinos
/
Animais Geneticamente Modificados
/
Transgenes
/
Técnicas de Transferência Nuclear
Tipo de estudo:
Diagnostic_studies
Limite:
Animals
Idioma:
En
Revista:
Genet. mol. res. (Online)
Assunto da revista:
BIOLOGIA MOLECULAR
/
GENETICA
Ano de publicação:
2005
Tipo de documento:
Article
País de afiliação:
Brasil
País de publicação:
Brasil