Science · 1997 · 905 citations · 20 references
Life SciencesBiotechnologyGeneticsEmbryo TransferGene ExpressionGenetic EngineeringEmbryo Transfer TechnologyPronuclear MicroinjectionSomatic Cell Nuclear TransferTransgenesis StrategiesTransgenic Animal TechnologyTransgenic Organism ProductionCloning By Somatic Cell Nuclear TransferManipulationMarker Gene IntegrationMicroinjectionNuclear Transfer EmbryologyNuclear Transfer TechniquesTransgenic Animal GenerationTransgenic Sheep
Nuclear transfer for generating transgenic sheep uses less than half the animals required for pronuclear microinjection. Ovine fetal fibroblasts were cotransfected with a neomycin resistance marker and a human factor IX construct, and the resulting cloned transfectants and G418‑resistant cell population were used as nuclear donors for transfer into enucleated oocytes. Six transgenic lambs were born; those derived from cloned cells carried both factor IX and neo transgenes, while those from the uncloned population carried only the marker, demonstrating that somatic cells can be genetically manipulated in vitro to produce viable animals by nuclear transfer.
Ovine primary fetal fibroblasts were cotransfected with a neomycin resistance marker gene (neo) and a human coagulation factor IX genomic construct designed for expression of the encoded protein in sheep milk. Two cloned transfectants and a population of neomycin (G418)-resistant cells were used as donors for nuclear transfer to enucleated oocytes. Six transgenic lambs were liveborn: Three produced from cloned cells contained factor IX and neo transgenes, whereas three produced from the uncloned population contained the marker gene only. Somatic cells can therefore be subjected to genetic manipulation in vitro and produce viable animals by nuclear transfer. Production of transgenic sheep by nuclear transfer requires fewer than half the animals needed for pronuclear microinjection.
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Sheep cloned by nuclear transfer from a cultured cell line
Keith Campbell, J. McWhir, William A. Ritchie et al. · Nature · 1996 · 1.8K citations
Agriculture, Cell Biology, Genetics +11
Production of transgenic rabbits, sheep and pigs by microinjection
Robert E. Hammer, V. G. Pursel, Caird E. Rexroad et al. · Nature · 1985 · 1.1K citations