| Project/Area Number |
10470173
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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 | 一般 |
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| Research Field |
Pediatrics
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| Research Institution | TOHOKU UNIVERSITY |
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Principal Investigator |
TSUCHIYA Shigeru Institute of Development, Aging, and Cancer, Tohoku University, Professor, 加齢医学研究所, 教授 (30124605)
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| Co-Investigator(Kenkyū-buntansha) |
KUMAKI Satoru Institute of Development, Aging, and Cancer, Tohoku University, Instructor, 加齢医学研究所, 助手 (20311566)
OHASHI Yoshiyuki Institute of Development, Aging, and Cancer, Tohoku University, Instructor, 加齢医学研究所, 助手 (60250825)
MINEGISHI Masayoshi Institute of Development, Aging, and Cancer, Tohoku University, Associate Professor, 加齢医学研究所, 助教授 (20211592)
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| Project Period (FY) |
1998 – 1999
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| Project Status |
Completed (Fiscal Year 1999)
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| Budget Amount *help |
¥11,700,000 (Direct Cost: ¥11,700,000)
Fiscal Year 1999: ¥3,700,000 (Direct Cost: ¥3,700,000)
Fiscal Year 1998: ¥8,000,000 (Direct Cost: ¥8,000,000)
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| Keywords | Antifection / Anti-CD3 antibody / pEGFP / WASP遺伝子野性型 / WASP遺伝子変異型 / GFP融合蛋白 / フローサイトメーター |
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| Research Abstract |
Purpose : To develop an antifection method, a new technique of antibody-mediated targeted gene transfection, we determined optimal conditions of the gene transfer.
Materials and Methods : OKT3, the antibody (Ab) against CD3 was chemically conjugated using benzoquinone to pEGFP, plasmid DNA containing green fluorescent protein (GFP) gene. The Ab-DNA conjugate was enriched by layering the mixture on a 40% sucrose solution followed by an ultracentrifugation. Coupling between DNA and Ab was determined by a 1% agarose gel electrophoresis followed by western blotting by anti-mouse IgG antibody. Jurkat T cells expressing CD3 were incubated with the Ab-DNA conjugate, and then determined the expression of GFP by flowcytometry.
Results and Discussion : Agarose gel electrophoresis showed a change of the electrophoretic mobility in the Ab-DNA conjugate, as compared to plasmid DNA alone. The western blot revealed that anti-mouse IgG antibody reacted against the observed band in the Ab-DNA reaction mixture, indicating the existence of the Ab-DNA conjugate. Successful transfection of the Ab-DNA conjugate into Jurkat cells was observed by coupling lipids (FuGENE) to the Ab-DNA conjugate. Chloroquine failed to confer successful transfection due to its cytotoxicity. Antifection could be an useful method for gene therapy.
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