Cellular and Molecular Biotechnology Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), 2-4-7 Aomi, Koutou-ku, Tokyo, 135-0064, Japan.
RIKEN Center for Biosystems Dynamics Research (BDR), Kobe, Hyogo, 650-0047, Japan.
Sci Rep. 2024 Sep 2;14(1):20327. doi: 10.1038/s41598-024-71105-y.
In female eutherian mammal development, X-chromosome inactivation (XCI) of one of the two X chromosomes is initiated early. Understanding the relationship between the initiation of XCI and cell fate is critical for understanding early female development and requires a system that can monitor XCI in single living cells. Traditional embryonic stem cells (ESCs) used for XCI studies often lose X chromosomes spontaneously during culture and differentiation, making accurate monitoring difficult. Additionally, most XCI assessment methods necessitate cell disruption, hindering cell fate tracking. We developed the Momiji (version 2) ESC line to address these difficulties, enabling real-time monitoring of X-chromosome activity via fluorescence. We inserted green and red fluorescent reporter genes and neomycin and puromycin resistance genes into the two X chromosomes of PGK12.1 ESCs, creating a female ESC line that retains two X chromosomes more faithfully during differentiation. Momiji (version 2) ESCs exhibit a more stable XX karyotype than other ESC lines, including the parental PGK12.1 line. This new tool offers valuable insights into the relationship between XCI and cell fate, improving our understanding of early female development.
在雌性真兽类哺乳动物的发育过程中，两条 X 染色体中的一条会较早启动 X 染色体失活（XCI）。了解 XCI 的启动与细胞命运之间的关系对于理解早期雌性发育至关重要，这需要一个能够在单个活细胞中监测 XCI 的系统。传统的用于 XCI 研究的胚胎干细胞（ESCs）在培养和分化过程中常常会自发丢失 X 染色体，这使得准确监测变得困难。此外，大多数 XCI 评估方法需要破坏细胞，从而阻碍了对细胞命运的跟踪。我们开发了 Momiji（版本 2）ESC 系来解决这些困难，通过荧光实现了对 X 染色体活性的实时监测。我们将绿色和红色荧光报告基因以及新霉素和嘌呤霉素抗性基因插入到 PGK12.1 ESCs 的两条 X 染色体中，创建了一条能够在分化过程中更忠实保留两条 X 染色体的雌性 ESC 系。Momiji（版本 2）ESC 系比包括亲本 PGK12.1 系在内的其他 ESC 系具有更稳定的 XX 核型。这个新工具为 XCI 与细胞命运之间的关系提供了有价值的见解，增进了我们对早期雌性发育的理解。
