Isolation of transfected fibroblast clones for use in nuclear transfer and transgene detection in cattle embryos.
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.
Embrapa Recursos Genéticos e Biotecnologia, Parque Estação Ecológica, Final W5, Asa Norte, 70770-900 Brasília, DF, Brasil. eom@cenargen.embrapa.br
Eduardo O
EO
Melo
Regivaldo V
RV
Sousa
Lílian T
LT
Iguma
Maurício M
MM
Franco
Elibio L
EL
Rech
Rodolfo
R
Rumpf
D000818:Animals; D030801:Animals, Genetically Modified / Q000196:embryology / Q000235:genetics*; D002417:Cattle / Q000196:embryology / Q000235:genetics*; D002467:Cell Nucleus / Q000235:genetics; D005347:Fibroblasts / Q000166:cytology; D053652:Nuclear Transfer Techniques; D019076:Transgenes / Q000235:genetics*