2004 Fiscal Year Final Research Report Summary
Development of method for gene function analysis using lentiviral vectors
| Project/Area Number |
15510167
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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 |
Applied genomics
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| Research Institution | RIKEN |
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Principal Investigator |
MIYOSHI Hiroyuki RIKEN, Technology and Development team for Biosignal Program, Subteam Leader, 生体情報統合技術開発チーム, サブチームリーダー (70219830)
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| Project Period (FY) |
2003 – 2004
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| Keywords | lentivirus / vector / gene transfer / gene function analysis / siRNA |
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| Research Abstract |
With the complete sequencing of the human and mouse genomes, the interest in tools for efficient gene function analysis has increased. Lentiviral vectors have been developed for gene therapy targeting nondividing cells. In this study, we have made several modifications to lentiviral vectors and developed method for basic research. We have also analyzed function of some genes using lentiviral vectors in collaboration with other laboratories.
1.We have developed a third generation lentiviral vector to increase safety and transduction efficiencies. This lentiviral vector has been shown to be capable of efficient gene transfer into hematopoietic stem cells, resting primary CD4^+ or CD8^+ T cells, B cells, NK cells, macrophages, human leukemia cells, and melanoma cells. Using lentiviral vectors, CD226 (DNAM-1) gene in CD4^+ naive T cells, Bcl-3 in macrophages, a chimeric immunoglobulin T cell receptor gene in T cells, and gp34/OX40L gene in dendritic cells have been analyzed.
2.We have constructed cre-lox vectors and tet-inducible vectors to control transgene expression.
3.We have developed siRNA(small interfering RNA) expressing lentiviral vectors for downregulating gene expression. Using siRNA expressing vectors, inactivation of PPARγ gene, mutated BRAF gene, Skp-2 gene, etc.facilitated the functional characterization of each gene. In order to control siRNA expression, we have constructed lentiviral vectors containing the tetracycline operator site in the Hi promoter and the tetracycline represser gene.
4.We have introduced cDNA library into lentiviral vectors and established expression cloning system. This system allowed us to identify genes that render cells resistant to HIV-induced cell death.
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