Geminin represses mesendoderm cell fate acquisition in embryoid bodies

Geminin is a small nucleoprotein that neuralizes ectoderm in the Xenopus embryo. Geminin promotes neural fate acquisition of mouse embryonic stem cells: Geminin knockdown during neural fate acquisition decreased expression of neural precursor cell markers (Pax6, Sox1), while increasing expression of Pitx2, Lefty1 and Cited2, genes involved in formation of the mouse node. Here we differentiated mouse embryonic stem cells into embryoid bodies to study Geminin's ability to repress primitive streak mesendoderm fate acquisition. We used microarrays to define the sets of genes that are regulated by Geminin during cell fate acquisition in embryoid bodies, using Dox-inducible Geminin knockdown or overexpression mouse embryonic stem cell lines. ES cell lines for Geminin over-expression (GemOE) were treated without or with Dox from day 3 to day 5 of EB differentiation and were collected on days 4 or 5 for microarray analysis. Gem knockdown (KD) ES cell lines were treated without or with Dox from day 0 to day 4 of EB differentiation and were collected on day 4 for microarray analysis.

Geminin是一种小型核蛋白，可在爪蟾（Xenopus）胚胎中介导外胚层神经化。Geminin可促进小鼠胚胎干细胞的神经命运获得：在神经命运获得过程中敲低Geminin，会降低神经前体细胞标志物（Pax6、Sox1）的表达水平，同时升高参与小鼠原结形成的基因Pitx2、Lefty1及Cited2的表达。本研究通过将小鼠胚胎干细胞诱导分化为胚状体（embryoid bodies，EB），探究Geminin抑制原条中内胚层命运获得的功能。我们构建了Dox诱导型Geminin敲低或过表达的小鼠胚胎干细胞系，并利用该细胞系在胚状体分化过程中通过基因芯片分析，确定受Geminin调控的细胞命运相关基因集。针对Geminin过表达（GemOE）的干细胞系，在胚状体分化的第3天至第5天分别施加或不施加Dox处理，并在第4天或第5天收集样本用于基因芯片分析。针对Geminin敲低（KD）的干细胞系，在胚状体分化的第0天至第4天分别施加或不施加Dox处理，并在第4天收集样本用于基因芯片分析。