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

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.

为了研究不同造血谱系中分化和可能的谱系转换或转分化的分子机制，我们生成了可诱导形式的骨髓转录因子 C/EBP α 和 PU.1，并在体外或体内检查了它们的全长效果。 C/EBP a 和 PU.1 与雌激素受体的配体结合域（C/EBP α -ER 和 PU.1-ER）融合在框内，以便它们可以被 4-羟基他莫昔芬激活(4-HT)。我们将 C/EBP α-ER 和 PU.1-ER 亚克隆到逆转录病毒载体、pMX-IRES-GFP 中，并感染病毒至 BaF3 细胞，BaF3/PU.1l-ER 细胞诱导生长停滞。细胞凋亡，表明 C/EBP α -ER 和 PU.1-ER 正常工作。 接下来我们亚克隆 Cl EBP α -ER 和。 PU.1-ER在H-2K启动子载体中获得了转基因小鼠的C/EBP a-ER和5个品系的转基因整合，RT-PCR分析显示mRNA在5个品系中表达。在 8 个 C/EBP α -ER 转基因株系中，通过蛋白质印迹仅 1 个株系表达 C/EBP α -ER 蛋白，C/EBP α -ER 在胸腺和脾脏中高表达，在骨髓和骨髓中中度表达。外周血凝胶转移测定表明，C/EBP α-ER 蛋白响应 4-HT 与保守的 C/EBP 结合序列结合，我们现在计划研究如何异位诱导 C/EBP。 α活性影响造血细胞在各个发育阶段的分化。

项目成果

Carpino N.: "Identification, cDNA cloning, and targeted deletion of p70, a novel, ubiquitously expressed SH3 domain-containing protein."Mol.Cell.Biol.. 22. 7491-7500 (2002)

Carpino N.：“p70 的鉴定、cDNA 克隆和靶向删除，p70 是一种新型、普遍表达的含有 SH3 结构域的蛋白质。”Mol.Cell.Biol.. 22. 7491-7500 (2002)

Carpino N., Kobayashi R., Zang H., Takahashi Y., Jou ST., Feng J., Nakajima H., Ihle JN.: "Identification, cDNA cloning, and targeted deletion of p70, a novel, ubiquitously expressed SH3 domain-containing protein."Mol.Cell.Biol.. 22. 7491-7500 (2002)

Carpino N.、Kobayashi R.、Zang H.、Takahashi Y.、Jou ST.、Feng J.、Nakajima H.、Ihle JN.：“p70（一种新型、普遍表达的 SH3）的鉴定、cDNA 克隆和靶向删除

Kitamura T., Koshino Y., Shibata F., Oki T., Nakaiima H., Nosaka T., Kumagai H.: "Retrovirus-mediated gene transfer and expression cloning : powerful tools in functional genomics."Exp.Hematol.. 31. 1007-1014 (2003)

Kitamura T.、Koshino Y.、Shibata F.、Oki T.、Nakaiima H.、Nosaka T.、Kumagai H.：“逆转录病毒介导的基因转移和表达克隆：功能基因组学的强大工具。”Exp.Hematol..

Awaya N.: "Novel variant isoform of G-CSF receptor involved in inductioin of proliferation of FDCP-2 cells : relevance to the pathogenesis of myelodysplastic syndrome."J.Cell.Physiol.. 191. 327-335 (2002)

Awaya N.：“参与 FDCP-2 细胞增殖诱导的 G-CSF 受体新变体亚型：与骨髓增生异常综合征发病机制的相关性。”J.Cell.Physiol.. 191. 327-335 (2002)

Watanabe N.: "Functional phenotype of phosphoinositide 3-kinase p85 alpha-null platelets characterized by impaired response to GP VI stimulation."Blood. 102. 541-548 (2003)

Watanabe N.：“磷酸肌醇 3-激酶 p85 α 缺失血小板的功能表型，其特征是对 GP VI 刺激的反应受损。”血液。