Analysis of hematopoietic lineage plasticity by using inducible transcription factors

利用诱导转录因子分析造血谱系可塑性

• 批准号: 14370298
• 负责人: NAKAJIMA Hideaki
• 金额: $ 8.9万
• 依托单位: The University of Tokyo
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B)
• 财政年份: 2002
• 资助国家: 日本
• 起止时间: 2002 至 2003
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-14370298/
• 关键词: differentiation; transcription factor; C; EBPα; PU.1; transigenic mouse; hematopoiesis; EBPε; differentiation; transcription factor; C; EBPα; PU.1; transigenic mouse; hematopoiesis; EBPε

In order to investigate the molecular mechanism of differentiation and possible lineage switch or trans-differentiation in various hematopoietic lineages, we generated inducible form of myeloid transcription factors C/EBP α and PU.1 and examined their effect in vitro or in vivo. Full length C/EBP a and PU.1 were fused in frame with ligand binding domain of estrogen receptor (C/EBP α -ER and PU.1-ER) so that they can be activated by 4-hydroxy tamoxifen (4-HT). We subcloned. C/EBP α -ER and PU.1-ER into retrovirus vector, pMX-IRES-GFP and infected virus to BaF3 cells. Interestingly, 4-HT treatment of BaF3/CEBP α -ER, BaF3/PU.1l-ER cells induced growth arrest and apoptosis, indicating that C/EBP α -ER and PU.1-ER are working properly. Next we subcloned Cl EBP α -ER and PU.1-ER in H-2K promoter vector made transgenic mice. We obtained transgene integration in 8 lines for C/ EBP a -ER and 5 lines for PU.1-ER. RT-PCR analysis showed that mRNA is expressed in 5 out of 8 C/EBP α -ER transgenics and only one line expressed C/EBP α -ER protein by western blot. C/EBP α -ER was expressed highly in thymus and spleen, moderately in bone marrow and peripheral blood. Gel-shift assay showed that C/ EBP α -ER protein bound to conserved C/EBP binding sequence in response to 4-HT. With these mice in our hand, we are now planning to investigate how ectopically induced C/ EBP α activity affects differentiation of hematopoietic cells at various developmental stages.

为了研究各种造血谱系中分化和可能的谱系开关或跨分化的分子机制，我们产生了髓样转录因子C/EBPα和PU.1的诱导形式，并在体外或体内检查了它们的作用。将全长C/EBP A和PU.1与雌激素受体的配体结合结构域（C/EBPα-ER和PU.1-ER）融合在一起，以便可以通过4-羟基他莫昔芬（4-HT）激活它们。我们亚克隆。 C/EBPα-ER和PU.1-ER进入逆转录病毒载体，PMX-IRES-GFP以及对BAF3细胞的感染病毒。有趣的是，BAF3/CEBPα-ER的4-HT治疗，BAF3/PU.1L-ER细胞诱导生长停滞和凋亡，表明C/EBPα-ER和PU.1-ER正在正常工作。接下来，我们在H-2K启动子载体中亚克隆的ClEBPα-ER和PU.1-ER制成了转基因小鼠。我们在C/EBP A -ER的8行中获得了翻译集成，而PU.1 -ER的5行。 RT -PCR分析表明，在8 C/EBPα-ER转换中，有5个表示mRNA，只有一条线通过蛋白质印迹表达C/EBPα-er蛋白。 C/EBPα-ER在胸腺和脾脏中高度表达，在骨髓和外周血中适度。凝胶转移测定表明，C/EBPα-ER蛋白构成响应4-HT的C/EBP结合序列。在我们手中的这些小鼠的情况下，我们现在正计划研究环保诱导的C/EBPα活性如何影响各个发育阶段的造血细胞的分化。