Primary mouse embryonic fibroblasts

In vitro studies with unmodified and modified N-terminal peptides of H3 demonstrated that Lys-14 acetylation did not interfere with methylation at Lys-9 by Suv39hl, while phosphorylation at Ser-10 and acetylation at Lys-9 did (Fig. 7). Further dimethylation of Lys-9 reduced enzymatic activity [186], A Suv39h double null primary mouse embryonic fibroblasts had higher levels of Ser-10 phos-phorylated H3 than wild type cells. These mutant cells had increased numbers of micro- and polynuclei. Oversized nuclei were characteristic of subpopulation of cells. The level of Lys-9 methylated FI3 in wild type cells and Suv39h double null cells was similar, demonstrating that other FI3 methyltransferases were involved [195]. Phosphorylation of Ser-10 by Ipll/aurora was also studied. Acetylation at Lys-14 promoted the activity of the mitotic kinase, while dimethylation, but not acetylation at Lys-9, reduced activity of the kinase [186]. 

Primary mouse embryonic fibroblast (EMFI) cells (37) or the STO fibroblast cell line (38) is the most commonly used feeder layers. Details for the preparation of a stock of EMFI or STO cells are described elsewhere (39). A brief protocol for the preparation of EMFI feeder layers will be given here. 

Mouse E14 ES cells were routinely grown onto a monolayer of mitomycin-treated primary embryonic mouse fibroblasts as a source of cytokines and growth factors. The medium consisted of DMEM with 15% fetal calf serum (Gibco, Cergy Pontoise, France), 100 U/mL penicillin, 100 pg/mL streptomycin, 1 pM a-mercaptoe-thanol, and 10 U/mL leukemia-inhibitory factor (LIF Gibco). ES cells were grown at 37°C in a humidified chamber with 10% CO2. ES cells were recovered with trypsin treatment for SdFFF experiments or subcultures. The SdFFF separation device used in this study includes a separation channel, which consisted of two 870 x 30 x... 

The work was carried out on the primary culture of embryonic tissues — the mouse and human fibroblasts [16, 17], We analyzed chromosomal aberrations in cells at the stages of anaphase or telophases (ana-telophases), after treatment culture by phosphemid in a concentration of 1 x 10 W. 

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