| Project/Area Number |
17590990
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| Research Category |
Grant-in-Aid for Scientific Research (C)
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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 | Chubu University (2006)
Nagoya University (2005) |
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Principal Investigator |
ASANO Haruhiko Chubu University, Department of Biomedical Sciences, Associate Professor, 生命健康科学部, 准教授 (10378060)
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| Project Period (FY) |
2005 – 2006
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| Project Status |
Completed (Fiscal Year 2006)
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| Budget Amount *help |
¥3,300,000 (Direct Cost: ¥3,300,000)
Fiscal Year 2006: ¥1,700,000 (Direct Cost: ¥1,700,000)
Fiscal Year 2005: ¥1,600,000 (Direct Cost: ¥1,600,000)
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| Keywords | fetal globin / transcription factor / zinc finger / gene therapy / luciferase / ZFF29 / 強制発現系 / Tet Onシステム / 遺伝子発現 |
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| Research Abstract |
To test the activity of ZFF29 proteins on the β-like globin gene transcription in non-erythroid cells, expression vectors of ZFF29 genes were constructed and transduced into non-erythroid lines, HL60 and KG-1. mRNA expression of globin genes in G418-resistant cells were analyzed by RT-PCR. Forced expression of ZFF29 genes failed to increase or induce globin gene transcription in neither type of cell lines. It was interesting, however, that ε-globin gene was constitutively expressed in KG-1 cells but not in HL60 cells.
Molecular mechanisms underlying transcriptional control of FKLF-2 (KLF13) gene expression in erythroid cells were analyzed by using erythroid (K562) cells and non-erythroid (COS7) cells. First of all we cloned upstream region of FKLF-2 gene by inverse PCR, and determined transcription initiation site by primer extension analysis. Sequential deletion mutants by restriction enzymes revealed that a 370 bp sequence to the transcription initiation site was necessary for the full promoter activity. Features of the FKLF-2 promoter were that 1)the promoter activity was two times more active in K562 cells than in COS7 cells ; 2)GATA-1 was potential trans activator of the promoter. Our results may shed lights on the molecular control of the erythroid cell development.
To test whether FKLF (KLF11) can activate γ-globin gene expression in adult erythroid cells, FKLF gene was introduced into MEL cells and mice carrying human γ-globin gene as a trans-gene. Frequency and amount of γ globin gene expressed in the MEL cells or the transgenic mouse were enhanced upon induction of FKLF trans-gene, while FKLF failed to activate the silent γ globin gene in the environment of adult erythropoiesis. These results suggest that transduction of FKLF by retrovirus vector may be used for the purpose of treatment of patients with sickle cell disease or β-thalassemia.
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