| Project/Area Number |
14370298
|
|---|
| Research Category |
Grant-in-Aid for Scientific Research (B)
|
| Allocation Type | Single-year Grants |
|---|
| Section | 一般 |
|---|
| Research Field |
Hematology
|
|---|
| Research Institution | The University of Tokyo |
|---|
Principal Investigator |
NAKAJIMA Hideaki The University of Tokyo, Institute of Medical Science, Research associate, 医科学研究所, 助手 (30217723)
|
|---|
| Co-Investigator(Kenkyū-buntansha) |
KITAMURA Toshio The University of Tokyo, Institute of Medical Science, Professor, 医科学研究所, 教授 (20282527)
|
|---|
| Project Period (FY) |
2002 – 2003
|
| Project Status |
Completed (Fiscal Year 2003)
|
| Budget Amount *help |
¥13,900,000 (Direct Cost: ¥13,900,000)
Fiscal Year 2003: ¥6,700,000 (Direct Cost: ¥6,700,000)
Fiscal Year 2002: ¥7,200,000 (Direct Cost: ¥7,200,000)
|
|---|
| Keywords | differentiation / transcription factor / C / EBPα / PU.1 / transigenic mouse / hematopoiesis / EBPε |
|---|
| 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.
|
