1992 Fiscal Year Final Research Report Summary
Investigation on gene therapy for congenital bleeding tendency
| Project/Area Number |
03454523
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| Research Category |
Grant-in-Aid for General Scientific Research (B)
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| Allocation Type | Single-year Grants |
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| Research Field |
Hematology
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| Research Institution | Nagoya University |
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Principal Investigator |
SAITO Hidehiko Nagoya University School of Medicine, Professor, 医学部, 教授 (20153819)
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| Co-Investigator(Kenkyū-buntansha) |
EMI Nobuhiko Nagoya University School of Medicine, Senior Resident, 医学部, 医員
TANIMOTO Mitsune Nagoya University School of Medicine, Assistant Professor, 医学部, 助手 (10240805)
TAKAMATSU Junki Nagoya University School of Medicine, Associate Professor, 医学部, 助教授 (80221365)
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| Project Period (FY) |
1991 – 1992
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| Keywords | factor IX / expression vector / retroviral vector / pL9RNL / gene therapy |
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| Research Abstract |
We constructed a new factor IX expression vector containing a full length factor IX cDNA.The Moloney Murine Leukemia Virus (MoMLV)-based retroviral vector pLRNL was cloned with the entire coding region of human factor IX down stream of the promoter of Rous sarcoma virus, givinh rise to the construct pL9RNL.The GP+E 86 packaging cell was transfected with pL9RNL and two mouse fibroblast cell lines (PA317 and NIH3T3) were infected with the virus generated from PA317 cell. During the 24 hr culture period, the maximum 1.2mug of factor IX was secreted into the medium from 10^6 of GP+E 86 cells. Factor IX in the culture media had the relatively normal procoagulant activity and was barium-citrate precipitated. Western blotting analysis of the barium-citrate precipitated revealed that a single chain 50Kd protein indistinguishable from the plasma-derived factor IX was produced in these three cells. The retroviral expression system that we utilized herein may contribute to the study of recombinant wild type or various mutant factor IX,and to the basic study for the future gene therapy.
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