title: A CO-FISH assay to assess sister chromatid segregation patterns in mitosis of mouse embryonic stem cells.
abstract: Sister chromatids contain identical DNA sequence but are chiral with respect to both their helical handedness and their replication history. Emerging evidence from various model organisms suggests that certain stem cells segregate sister chromatids nonrandomly to either maintain genome integrity or to bias cellular differentiation in asymmetric cell divisions. Conventional methods for tracing of old vs. newly synthesized DNA strands generally lack resolution for individual chromosomes and employ halogenated thymidine analogs with profound cytotoxic effects on rapidly dividing cells. Here, we present a modified chromosome orientation fluorescence in situ hybridization (CO-FISH) assay, where identification of individual chromosomes and their replication history is achieved in subsequent hybridization steps with chromosome-specific DNA probes and PNA telomere probes. Importantly, we tackle the issue of BrdU cytotoxicity and show that our method is compatible with normal mouse ES cell biology, unlike a recently published related protocol. Results from our CO-FISH assay show that mitotic segregation of mouse chromosome 7 is random in ES cells, which contrasts previously published results from our laboratory and settles a controversy. Our straightforward protocol represents a useful resource for future studies on chromatid segregation patterns of in vitro-cultured cells from distinct model organisms.
authors.affiliation: Gene Regulation and Chromosome Biology Laboratory, Frederick National Laboratory for Cancer Research, National Institutes of Health, Frederick, MD 21702, USA.
authors.forename: Stephan
authors.initials: S
authors.lastname: Sauer
authors.forename: Sandra S
authors.initials: SS
authors.lastname: Burkett
authors.forename: Mark
authors.initials: M
authors.lastname: Lewandoski
authors.forename: Amar J S
authors.initials: AJ
authors.lastname: Klar
mesh_terms: D000818:Animals; D001973:Bromodeoxyuridine / Q000378:metabolism / Q000633:toxicity; D002470:Cell Survival / Q000187:drug effects; D002842:Chromatids / Q000378:metabolism*; D020090:Chromosome Segregation / Q000187:drug effects; D033481:Chromosomes, Mammalian / Q000378:metabolism; D053595:Embryonic Stem Cells / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism*; D017404:In Situ Hybridization, Fluorescence / Q000379:methods*; D051379:Mice; D008938:Mitosis / Q000187:drug effects
chemical_list: D001973:Bromodeoxyuridine