Mouse embryonic fibroblast (MEF) derivation and FISH analyses

Primary MEFs were prepared from F1 E13.5 mouse embryos (129S4 x C57BL/6N, where the father was homozygous for the Oct4:EGFP transgene). Briefly, the embryo body cavity was minced using sterile blades, and a single cell suspension obtained using 0.25% Trypsin for 15 min. The cell mix was then thoroughly dissociated in MEF media, consisting of DMEM with 10% fetal calf serum, 0.1 M nonessential amino acids, 2 mM L-glutamine, and penicillin/streptomycin (each from Thermo Fisher Scientific, Waltham MA). Cells were then cultured until confluent.

FISH probes were generated from a BAC specific to Chromosome 9 (RP24-402I19), and from a plasmid containing the Oct4:EGFP transgene (GOF18ΔPE EGFP, Addgene plasmid #52382; RRID:Addgene_52382).

FISH staining was performed on MEF cells as previously described (Saxena et al. 2000). Cells were harvested by trypsinization, treated with 0.075 M KCl hypotonic solution and fixed in methanol:acetic acid (3:1 v/v). The cell suspension was applied to slides and treated with RNase. After dehydration in ethanol, slides were incubated at 80° for 5 min to denature the DNA and washed again in ethanol.

BAC probes were labeled by nick translation: the Cy-3 probe is fluorescent, and Biotin probe was subsequently detected by avidin conjugated with FITC (#434411, ThermoFisher Scientific). The probes were denatured together with unlabeled competitor DNA and applied to the slides. Slides with probes were incubated at 37° for 16 h, washed in 50% formamide to remove non-specific hybridization, and dehydrated in ethanol. Chromosomes were stained with DAPI and imaged on a fluorescence microscope.