Molecular Genetics of Stem Cells Laboratory, Institute of Research in Immunology and Cancer, University of Montreal, Montreal, Quebec H3C 3J7, Canada.
Cell Stem Cell. 2010 Jul 2;7(1):101-13. doi: 10.1016/j.stem.2010.06.007.
In this study, we describe an in vivo RNA interference functional genetics approach to evaluate the role of 20 different conserved polarity factors and fate determinants in mouse hematopoietic stem cell (HSC) activity. In total, this screen revealed three enhancers and one suppressor of HSC-derived reconstitution. Pard6a, Prkcz, and Msi2 shRNA-mediated depletion significantly impaired HSC repopulation. An in vitro promotion of differentiation was observed after the silencing of these genes, consistent with their function in regulating HSC self-renewal. Conversely, Prox1 knockdown led to in vivo accumulation of primitive and differentiated cells. HSC activity was also enhanced in vitro when Prox1 levels were experimentally reduced, identifying it as a potential antagonist of self-renewal. HSC engineered to overexpress Msi2 or Prox1 showed the reverse phenotype to those transduced with corresponding shRNA vectors. Gene expression profiling studies identified a number of known HSC and cell cycle regulators as potential downstream targets to Msi2 and Prox1.
在这项研究中，我们描述了一种体内 RNA 干扰功能遗传学方法，用于评估 20 种不同的保守极性因子和命运决定因子在小鼠造血干细胞 (HSC) 活性中的作用。总的来说，该筛选揭示了三个增强子和一个抑制子对 HSC 衍生重建的作用。Pard6a、Prkcz 和 Msi2 shRNA 介导的耗竭显著损害了 HSC 的再定植。这些基因沉默后观察到体外分化的促进，与其在调节 HSC 自我更新中的功能一致。相反，Prox1 的敲低导致体内原始和分化细胞的积累。当 Prox1 水平通过实验降低时，HSC 的体外活性也得到增强，这表明它是自我更新的潜在拮抗剂。过表达 Msi2 或 Prox1 的 HSC 表现出与转导相应 shRNA 载体相反的表型。基因表达谱研究确定了一些已知的 HSC 和细胞周期调节剂作为 Msi2 和 Prox1 的潜在下游靶点。
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