Evaluation of gene targeting by homologous recombination in ovine somatic cells.
Mouse models for some human genetic diseases are limited in their applications since they do not accurately reproduce the phenotype of the human disease. It has been suggested that larger animals, for example sheep, might produce more useful models, as some aspects of sheep physiology and anatomy are more similar to those of humans. The development of methods to clone animals from somatic cells provides a potential novel route to generate such large animal models following gene targeting. Here, we assess targeting of the cystic fibrosis transmembrane conductance regulator (CFTR) gene in ovine somatic cells using homologous recombination (HR) of targeting constructs with extensive (>11 kb) homology. Electroporation of these constructs into ovine fetal and post-natal fibroblasts generated G418-resistant clones, but none analyzed had undergone HR, suggesting that at least for this locus, it is an extremely inefficient process. Karyotyping of targeted ovine fetal fibroblasts showed them to be less chromosomally stable than post-natal fibroblasts, and, moreover, extended culture periods caused them to senesce, adversely affecting their viability for use as nuclear transfer donor cells. These data stress the importance of donor cell choice in somatic cell cloning and suggest that culture time be kept to a minimum prior to nuclear transfer in order to maximize cell viability.
Williams
Sarah H
SH
Paediatric Molecular Genetics, Weatherall Institute of Molecular Medicine, Oxford University, John Radcliffe Hospital, Oxford, United Kingdom.
Sahota
Virender
V
Palmai-Pallag
Timea
T
Tebbutt
Scott J
SJ
Walker
Jean
J
Harris
Ann
A
D000818:Animals; D026501:Blotting, Southwestern; D002454:Cell Differentiation; D002478:Cells, Cultured; D016922:Cellular Senescence; D043171:Chromosomal Instability; D003001:Cloning, Molecular / Q000379:methods; D018389:Codon, Nonsense / Q000235:genetics; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000032:analysis / Q000235:genetics*; D004247:DNA / Q000235:genetics / Q000302:isolation & purification; D005333:Fetus / Q000166:cytology; D005347:Fibroblasts / Q000502:physiology*; D018390:Gene Targeting; D009154:Mutation; D016133:Polymerase Chain Reaction; D011995:Recombination, Genetic; D012756:Sheep / Q000196:embryology / Q000235:genetics; D019098:Telomerase / Q000378:metabolism; D014162:Transfection
D018389:Codon, Nonsense; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D004247:DNA; D019098:Telomerase