| Ornithine decarboxylase: Expression and regulation in rat brain and in transgenic mice | ||
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The ODC-deficient Chinese hamster ovary cell line, C55.7, was generously provided by Dr. I. Scheffler (University of California San Diego, La Jolla, CA, USA). C55.7 cells were grown in Dulbecco"s modified Eagle"s medium (DMEM, GibcoBRL, Paisley, UK) with low glucose and 10% fetal calf serum (FCS) supplemented with 0.5 mM putrescine, nonessential amino acids mixture and antibiotics. The cell lines derived by transfection of the C55.7 cells were grown in the above medium but without added putrescine. The cells expressing mutated mouse ODC were grown in a medium containing 0.1 µM dexamethasone in order to induce ODC-activity for maintaining constant growth.
The C55.7 cells were transfected using the standard calcium phosphate coprecipitation method with a glycerol shock (Ausubel et al. 1993). Two days after transfection, the cells were transferred to a medium lacking putrescine and containing dialyzed FCS for selection of the stable transfectants. The selected cultures were grown either in the presence or absence of 0.1 µM dexamethasone and selection was carried out for 17 - 21 days before isolating colonies. The integration of transfected constructs was confirmed both by the polymerase chain reaction and Southern analysis.
For ODC activity assays cells were fed with fresh medium containing 0.1 µM dexamethasone 7 hours before harvesting. Cells were homogenised by repeated freezing and thawing on a dry-ice bath and dissolved in 25 mM Tris-HCl buffer, pH 7.5, containing 2.5 mM DTT, 0.1 mM EDTA and 0.5 mM phenylmethylsulfonylfluoride (PMSF). For kinetic assays cell lysates were fractionated with a two-step (NH4)2SO4 precipitation (20 % - 60 %). The protein precipitate was dissolved in homogenisation buffer and dialysed before ODC activity assays.