Woodcock D M, Crowther P J, Simmons D L, Cooper I A
Exp Cell Res. 1986 Jan;162(1):23-32. doi: 10.1016/0014-4827(86)90423-4.
SCC30 cells (derived from a single cell from the Chinese hamster ovary CHO-K1 cell line, selected on the basis of a stable chromosome complement) were used to select cell variants with hypomethylated DNA. Cells were treated with 5-aza-2'-deoxycytidine (5azadCyd) at 0.1, 1, or 5 microM for two weeks with the medium and drug renewed twice weekly. From the few surviving cells, 25 random single cell-derived clones were grown for freezing cell stocks, and for DNA isolation for 5-methyldeoxycytidine (5medCyd) estimations. After a minimum of one month's recovery from the drug, these cells showed a continuum of 5medCyd levels ranging from ones with the same as the parental clone (2.93%) to ones having lost almost 50% of their DNA methylation. The modal value corresponded to a loss of one third to one quarter of methylated sites. Five subclones with hypomethylated DNA were grown from the frozen stocks. These cells were shown not to be 5azaCyd-resistant cell variants. By the time sufficient cells had been grown to determine DNA methylation levels, the average percentage of 5medCyd had increased to 76% of the SCC30 value compared to 67% at the time of freezing cell stocks. However, this level of DNA hypomethylation remained constant over two months of continuous culture. Cells of one of these hypomethylated subclones were subjected to a second cycle of 5azaCyd treatment. Six random clones from the survivors showed a further decrease averaging 11% in the level of DNA methylation but, by two months in continuous culture, 5medCyd levels had returned to that present before the second cycle of selection. Hence, cell variants can be readily obtained which have lost some 8-10 million methylated sites (pairs of methylated deoxycytidines), and this loss does not compromise cell viability in in vitro culture. This is consistent with mammalian genomes containing a high level of background methylation in non-essential sites. The usefulness of such single cell-derived clones with stably hypomethylated genomes is discussed in relation to understanding the functions of deoxycytidine methylation in mammalian DNA.
SCC30细胞（源自中国仓鼠卵巢CHO-K1细胞系的单个细胞，根据稳定的染色体互补性进行选择）用于筛选DNA低甲基化的细胞变体。细胞用0.1、1或5微摩尔的5-氮杂-2'-脱氧胞苷（5azadCyd）处理两周，每周更换两次培养基和药物。从少数存活细胞中，培养25个随机的单细胞衍生克隆用于冷冻细胞库，并用于分离DNA以进行5-甲基脱氧胞苷（5medCyd）测定。从药物处理中恢复至少一个月后，这些细胞的5medCyd水平呈现出一个连续范围，从与亲本克隆相同的水平（2.93%）到几乎失去50%DNA甲基化的水平。众数对应于甲基化位点损失三分之一到四分之一。从冷冻库中培养出五个DNA低甲基化的亚克隆。这些细胞被证明不是5azaCyd抗性细胞变体。当生长出足够数量的细胞以确定DNA甲基化水平时，5medCyd的平均百分比已增加到SCC30值的76%，而在冷冻细胞库时为67%。然而，这种DNA低甲基化水平在连续培养两个月期间保持恒定。其中一个低甲基化亚克隆的细胞接受了第二轮5azaCyd处理。幸存者中的六个随机克隆显示DNA甲基化水平进一步平均下降11%，但在连续培养两个月后，5medCyd水平已恢复到第二轮选择前的水平。因此，可以很容易地获得细胞变体，它们已经失去了约800万至1000万个甲基化位点（甲基化脱氧胞苷对），并且这种损失在体外培养中不会损害细胞活力。这与哺乳动物基因组在非必需位点含有高水平的背景甲基化是一致的。讨论了这种具有稳定低甲基化基因组的单细胞衍生克隆在理解脱氧胞苷甲基化在哺乳动物DNA中的功能方面的实用性。
