Tropel P, Roullot V, Vernet M, Poujol C, Pointu H, Nurden P, Marguerie G, Tronik-Le Roux D
Commissariat à l'Energie Atomique, Département de Biologie Moléculaire et Structurale, CEA-Grenoble, France.
Blood. 1997 Oct 15;90(8):2995-3004.
The continuous generation of mature blood cells from primitive multipotent progenitor cells requires a highly complex series of cellular events that are still largely unknown. To examine the molecular events associated with the commitment of these hematopoietic progenitor cells to the megakaryocytic lineage, the alpha subunit of the platelet integrin alphaIIb beta3 was used as marker. Despite an abundance of information regarding the role of this integrin in platelet adhesion and aggregation, the mechanisms that control the expression of the genes that code for these proteins are poorly understood and the earliest hematopoietic cell capable of expressing them has not been clearly identified. Thus, a strategy was developed to eradicate, using a conditional toxigene, all the hematopoietic cells capable of expressing the alphaIIb gene in mice. This was achieved by targeting the expression of the gene encoding the herpes simplex virus thymidine kinase (tk), specifically to these cell types, using a 2.7-kb fragment of the 5'-flanking region of the murine alphaIIb gene. Three transgenic lines having 1, 3, and 4 copies of the transgene, respectively were produced and analyzed. Administration of ganciclovir (GCV) to these mice induced a severe thrombocytopenia, which was due to the depletion of the entire megakaryocytic lineage, as shown by bone marrow (BM) culture and electron microscopy analysis. The time required to attain a severe thrombocytopenia was dependent on the level of the expression of the transgene and varied from 7 to 11 days. This condition was completely reversed when GCV treatment was discontinued. Progenitor cell assays showed that the alphaIIb promoter was active in primitive hematopoietic progenitor cells possessing myeloid, erythroid, and megakaryocytic potential and that the transcriptional activity of the promoter decreased progressively as differentiation proceeded towards the erythroid and myeloid lineages.
从原始多能祖细胞持续生成成熟血细胞需要一系列高度复杂的细胞事件，而这些事件在很大程度上仍不为人知。为了研究与这些造血祖细胞向巨核细胞谱系定向分化相关的分子事件，血小板整合素αIIbβ3的α亚基被用作标志物。尽管关于这种整合素在血小板黏附和聚集方面的作用已有大量信息，但控制编码这些蛋白质的基因表达的机制却知之甚少，而且能够表达它们的最早造血细胞尚未明确鉴定。因此，开发了一种策略，利用条件性毒基因清除小鼠体内所有能够表达αIIb基因的造血细胞。这是通过使用小鼠αIIb基因5'侧翼区的2.7 kb片段，将编码单纯疱疹病毒胸苷激酶（tk）的基因表达特异性靶向这些细胞类型来实现的。分别产生并分析了具有1个、3个和4个转基因拷贝的三个转基因品系。给这些小鼠施用更昔洛韦（GCV）会导致严重的血小板减少症，这是由于整个巨核细胞谱系的耗竭所致，骨髓（BM）培养和电子显微镜分析表明了这一点。达到严重血小板减少症所需的时间取决于转基因的表达水平，从7天到11天不等。当停止GCV治疗时，这种情况会完全逆转。祖细胞分析表明，αIIb启动子在具有髓系、红系和巨核系潜能的原始造血祖细胞中具有活性，并且随着向红系和髓系谱系的分化，启动子的转录活性逐渐降低。
