title: ES cell cycle rates affect gene targeting frequencies.
abstract: We have investigated the gene targeting frequency at the hprt locus in a range of embryonic stem cell lines selected for variations in cell cycle parameters. Our results show that targeting frequency varies with cell line by as much as 12-fold between nonisogenic lines and 3-fold between isogenic lines and that a nonisogenic line can support homologous recombination events by up to 21-fold more frequently than an isogenic line. This variation is consistent with both insertion and replacement vectors. These results can be explained by an inverse linear correlation of targeting frequencies with cell doubling times. Additionally, by reducing serum concentration in the culture medium the mean cell doubling time for R1 ES cells can be increased from 11.4 to 15.7 h, with a subsequent 15-fold decrease in gene targeting frequency. This change fits the correlation found for the different nonisogenic cell lines. Our observations have important implications when performing gene targeting experiments and explain some of the variation noted between experiments.
authors.affiliation: Molecular Embryology Group, AgResearch, Ruakura Research Centre, Hamilton, New Zealand. udyg@agresearch.cri.nz
authors.forename: G B
authors.initials: GB
authors.lastname: Udy
authors.forename: B D
authors.initials: BD
authors.lastname: Parkes
authors.forename: D N
authors.initials: DN
authors.lastname: Wells
mesh_terms: D000818:Animals; D002453:Cell Cycle; D002460:Cell Line; D004622:Embryo, Mammalian / Q000166:cytology; D018390:Gene Targeting; D007041:Hypoxanthine Phosphoribosyltransferase / Q000235:genetics; D051379:Mice; D008807:Mice, Inbred BALB C; D008815:Mice, Inbred Strains; D018345:Mice, Knockout; D015203:Reproducibility of Results; D013234:Stem Cells / Q000166:cytology*
chemical_list: D007041:Hypoxanthine Phosphoribosyltransferase