Six cloned calves produced from adult fibroblast cells after long-term culture

Cloning whole animals with somatic cells as parents offers the possibility of targeted genetic manipulations in vitro such as "gene knock-out" by homo logous recombination. However, such manipulation requires prolonged culture of nuclear donor cells. Previous successes in cloning have been limited to the use of cells collected either fresh or after short-term culture. There fore, demonstration of genetic totipotency of cells after prolonged culture is pivotal to combining site-specific genetic manipulations and cloning. H ere we report birth of six clones of an aged (17-year-old) Japanese Black B eef bull using ear skin fibroblast cells as nuclear donor cells after up to 3 months of in vitro culture (10-15 passages). We observed higher developm ental rates for embryos derived from later passages (10 and 15) as compared with those embryos from an early passage (passage 5). The four surviving c lones are now 10-12 months of age and appear normal, similar to their natur ally reproduced peers. These data show that fibroblasts of aged animals rem ain competent for cloning, and prolonged culture does not affect the clonin g competence of adult somatic donor cells.