Yoshida I, Nishita Y, Mohandas T K, Takagi N
Research Center for Molecular Genetics, Graduate School of Environmental Earth Science, Hokkaido University, Sapporo, Japan.
Exp Cell Res. 1997 Feb 1;230(2):208-19. doi: 10.1006/excr.1996.3393.
An inactive human X chromosome was introduced by microcell fusion into two mouse embryonal carcinoma cell lines, PSA1-TG8 and OTF9-63, each of which has a single X chromosome. The donor cell line was a mouse-human somatic cell hybrid, CF150, retaining one or more inactive human X chromosome(s) per cell as its only human element. Twenty hybrid clones isolated retained EC morphology and contained the intact human X chromosome(s) or its truncated derivative(s). Replication banding analysis showed that the introduced human X chromosome(s) or its derivative(s) replicated synchronously with other mouse chromosomes, suggesting reactivation of the human X chromosomal elements after transfer. Reversal of inactivation was further confirmed by the expression of five human X-linked genes repressed in CF150, although the XIST (X inactive specific transcript) gene continued to be active. The level of XIST expression in our hybrid cells was almost identical to that of parental CF150 cells. Methylation status of 5' end of the active XIST gene varied considerably from almost full methylation to unmethylation in these hybrids. Thus, mouse EC cells used in this study were capable of altering methylation status of the human XIST gene in a manner lacking consistency and unable to repress its transcription. Furthermore, we failed to obtain any positive evidence for the occurrence of X chromosome inactivation in differentiating monochromosome EC hybrids. Taken together, these findings suggest that the human X chromosome inactivation center including the XIST gene is unable to function effectively in mouse cells.
通过微细胞融合将一条失活的人类X染色体导入两种小鼠胚胎癌细胞系PSA1-TG8和OTF9-63，这两种细胞系均各有一条X染色体。供体细胞系是一种小鼠-人类体细胞杂种CF150，每个细胞保留一条或多条失活的人类X染色体作为其唯一的人类成分。分离得到的20个杂种克隆保持了胚胎癌细胞的形态，并含有完整的人类X染色体或其截短的衍生物。复制带型分析表明，导入的人类X染色体或其衍生物与其他小鼠染色体同步复制，提示转移后人类X染色体元件发生了重新激活。虽然XIST（X失活特异性转录本）基因仍保持活性，但在CF150中受抑制的5个人类X连锁基因的表达进一步证实了失活的逆转。我们杂种细胞中XIST的表达水平与亲本CF150细胞几乎相同。在这些杂种细胞中，活性XIST基因5′端的甲基化状态差异很大，从几乎完全甲基化到去甲基化。因此，本研究中使用的小鼠胚胎癌细胞能够以缺乏一致性且无法抑制其转录的方式改变人类XIST基因的甲基化状态。此外，我们未能获得任何关于在分化的单染色体胚胎癌杂种细胞中发生X染色体失活的阳性证据。综上所述，这些发现表明包括XIST基因在内的人类X染色体失活中心在小鼠细胞中无法有效发挥作用。
