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Isolation of transfected fibroblast clones for use in nuclear transfer and transgene detection in cattle embryos
Eduardo O. Melo1, Regivaldo V. Sousa1, Lílian T. Iguma1,2, Maurício M. Franco1,
Elibio L. Rech1 and Rodolfo Rumpf1
1Embrapa Recursos Genéticos e Biotecnologia, Parque Estação Ecológica,
Final W5, Asa Norte, 70770-900 Brasília, DF, Brasil
2Departamento de Biologia Celular, Universidade de Brasília,
70910-900 Brasília, DF, Brasil
Corresponding author: E.O. Melo
E-mail: eom@cenargen.embrapa.br
Genet. Mol. Res. 4 (4): 812-821 (2005)
Received April 3, 2005
Accepted July 12, 2005
Published December 27, 2005

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.

Key words: Cattle, Cell isolation, Multiplex-PCR, Nuclear transfer, Transgene detection

 

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