2003 Fiscal Year Final Research Report Summary
Analysis of hematopoietic lineage plasticity by using inducible transcription factors
| Project/Area Number |
14370298
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| Research Category |
Grant-in-Aid for Scientific Research (B)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Hematology
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| Research Institution | The University of Tokyo |
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Principal Investigator |
NAKAJIMA Hideaki The University of Tokyo, Institute of Medical Science, Research associate, 医科学研究所, 助手 (30217723)
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| Co-Investigator(Kenkyū-buntansha) |
KITAMURA Toshio The University of Tokyo, Institute of Medical Science, Professor, 医科学研究所, 教授 (20282527)
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| Project Period (FY) |
2002 – 2003
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| Keywords | differentiation / transcription factor / C / EBPα / PU.1 / transigenic mouse / hematopoiesis |
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| Research Abstract |
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.
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[Publications] 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)
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[Publications] Nosaka T., Morita S., Kitamura H., Nakajima H., Shibata F., Morikawa Y., Kataoka Y., Ebihara Y., Kawashima T., Itoh T., Ozaki K., Senba E., Tsuji K., Makishima F., Yoshida N., Kitamura T.: "Mammalian twisted gastrulation is essential for skeletolymphogenesis."Mol.Cell.Biol.. 23. 2969-2980 (2003)
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[Publications] Watanabe N., Nakajima H., Suzuki H., Oda A., Matsubara Y., Moroi M., Terauchi Y., Kadowaki T., Suzuki H., Koyasu S., Ikeda Y., Hanada M.: "Functional phenotype of phosphoinositide 3-kinase p85alpha-null platelets characterized by an impaired response to GP VI stimulation."Blood. 102. 541-548 (2003)
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[Publications] Murata K., Kumagai H., Kawashima T., Tamitsu K., Irie M., Nakajima H., Suzu S., Shibuya M., Kamihira S., Nosaka T., Asano S., Kitamura T.: "Selective cytotoxic mechanism of GTP-14564, a novel tyrosine kinase inhibitor in. leukemia cells expressing a constitutively active Fms-like tyrosine kinase 3 (FLT3)."J.Biol.Chem.. 278. 32892-32898 (2003)
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[Publications] Kumagai H., Oki T., Tamitsu K., Feng SZ., Ono M., Nakajima H., Bao YC., Kawakami Y., Nagayoshi K., Copeland NG., Gilbert DJ., Jenkins NA., Kawakami T., Kitamura T.: "Identification and characterization of a new pair of immunoglobulin-like receptors LMIR1 and 2 derived from murine bone marrow-derived mast cells."Biochem.Biophys.Res.Commun.. 307. 719-729 (2003)
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