title: Nuclear reprogramming of somatic cells by embryonic stem cells is affected by cell cycle stage.
abstract: Hybrid embryonic stem (ES)-like clones were generated by fusion of murine ES cells with somatic cells that carried a neo resistance gene under the transcriptional control of the Oct-4 promoter. The Oct-4 promoter was reactivated in hybrid ES cells formed by fusion with fetal fibroblasts, and all hybrid colonies were of ES rather than fibroblast phenotype, suggesting efficient reprogramming of fibroblast chromosomes. Like normal diploid murine ES cells, hybrid lines expressed alkaline phosphatase activity and formed differentiated cells derived from the three embryonic germ layers both in vitro and in vivo. Treatments thought to affect nuclear transfer efficiency (ES cell confluence and serum starvation of primary embryonic fibroblasts) were investigated to determine whether they had an effect on reprogramming in cell hybrids. Serum starvation of primary embryonic fibroblasts increased hybrid colony number 50-fold. ES cells were most effective at reprogramming when they contained a high proportion of cells in the S and G2/M phases of the cell cycle. These data suggest that nuclear reprogramming requires an initial round of somatic DNA replication of quiescent chromatin in the presence of ES-derived factors produced during S and G2/M phases.
authors.affiliation: Division of Gene Function and Development, Roslin Institute (Edinburgh), Midlothian, United Kingdom.
authors.forename: Stephen
authors.initials: S
authors.lastname: Sullivan
authors.forename: Steve
authors.initials: S
authors.lastname: Pells
authors.forename: Martin
authors.initials: M
authors.lastname: Hooper
authors.forename: Ed
authors.initials: E
authors.lastname: Gallagher
authors.forename: Jim
authors.initials: J
authors.lastname: McWhir
mesh_terms: D000469:Alkaline Phosphatase / Q000378:metabolism; D000818:Animals; D001483:Base Sequence; D002453:Cell Cycle / Q000502:physiology*; D002454:Cell Differentiation; D002460:Cell Line; D002467:Cell Nucleus / Q000235:genetics / Q000378:metabolism*; D016895:Culture Media, Serum-Free; D017931:DNA Primers / Q000235:genetics; D004261:DNA Replication; D005786:Gene Expression Regulation; D006822:Hybrid Cells / Q000166:cytology* / Q000378:metabolism; D051379:Mice; D010641:Phenotype; D039904:Pluripotent Stem Cells / Q000166:cytology* / Q000378:metabolism
chemical_list: D016895:Culture Media, Serum-Free; D017931:DNA Primers; D000469:Alkaline Phosphatase