1998 Fiscal Year Final Research Report Summary
New vector system for the gene therapy of abdominal cancers.
| Project/Area Number |
09557103
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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 | 展開研究 |
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| Research Field |
Digestive surgery
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| Research Institution | Nagoya University |
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Principal Investigator |
NIMURA Yuji Nagoya University School of Medicine Professor, 医学部, 教授 (80126888)
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| Co-Investigator(Kenkyū-buntansha) |
KANAI Michio Kasugai City Hospital Chief of the Surgical Department, 助手・外科部長 (50242871)
NAGINO Masato Nagoya University School of Medicine Associate Professor, 医学部, 講師 (20237564)
HAGIWARA Masatoshi Tokyo Medical and Dental University School of Medicine, Professor, 難治疾患研究所, 教授 (10208423)
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| Project Period (FY) |
1997 – 1998
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| Keywords | gene therapy / retroviral vector / peritoneal dessemination / transcriptional regulation |
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| Research Abstract |
Tight control of gene expression in temporal and quantitativemanner is an invaluable tool in both basic and clinical biology. The desired characteristics of the inducible system are as follows. (I) The regulator should be activated only upon administration of an exogenousmediator, and terminated when the exogenous stimulus is removed. (ii) The mediator should be non toxic and active upon oral administration. (iii) The system should not activate other endogenous cellular genes. We constricted a retroviral vector that has an autoregulatory cassette and express the gene of interest in response to oral administration of doxycycline (dox) invivo. The cassette contains the all components of the reversetetracycline-regulated (rtTA) system (Gossen M.et al. 1995), a drugselectable marker (BSD) with the internal ribosome entry site (IRES) and the gene of interest (GFP). The retroviral long term repeat enhancer and promoter elements driv-expression of the taransactivator (rTetR-VP16) and BSD, but
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the translation was terminated at the 3'-site of BSD. The expression of GFP was controlled under the tandem tet operator sequencesand the cytomegalovirus minimal promoter in a dose-dependent manner to dox. FACS analyses showed that GFP-fluoresence was induced in two-ordermagnitude in the retrovirus-infected 208F cells dependent on the amount of dox. Furthermore oral administration of dox could induce GFP protein in thetransplanted 208F cells in the peritoneal cavity of a nude mouse. Thus, this reverse tetracycline-regulated retroviral vector (RTRRV) system allowseasy delivery of controllable genes to cultured cells and transgenicanimals. However, the control system did not work in several human cancercells. This may be attributed to the species-dependent susceptibility toretorviruses. And we found that the introduction of a toxic gene into RTRRVkilled the PA317 retrovirus packaging cells, suggesting that RTRRV still suffer from problems of higher basal levels of gene expression undernoninduced condition and pleiotropic effects on host cell genes. If we will solve these problems mentions above, this inducible retroviral vector system may pave the way to the controlled gene expression during a particular window of time in gene therapy applications. Less
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