| Project/Area Number |
60440096
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| Research Category |
Grant-in-Aid for General Scientific Research (A)
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| Allocation Type | Single-year Grants |
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| Research Field |
医学一般
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| Research Institution | Osaka University |
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Principal Investigator |
UCHIDA Tsuyoshi Institute for Molecular and Cellular Biology, Osaka University, 細胞工学センター, 教授 (40029781)
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| Co-Investigator(Kenkyū-buntansha) |
ISHIURA Masahiro National Institute for Basic Biology, 基礎生物学研究所, 助手 (20132730)
YONEDA Yoshihiro Institute for Molecular and Cellular Biology, Osaka University, 細胞工学センター, 助手 (80191667)
MEKADA Eisuke Institute for Molecular and Cellular Biology, Osaka University, 細胞工学センター, 助手 (20135742)
KANEDA Yasufumi Institute for Molecular and Cellular Biology, Osaka University, 細胞工学センター, 助手 (10177537)
YAMAIZUMI Masaru Institute for Molecular and Cellular Biology, Osaka University, 細胞工学センター, 助教授 (70107093)
河野 憲二 大阪大学細胞工学センター (50142005)
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| Project Period (FY) |
1985 – 1987
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| Project Status |
Completed (Fiscal Year 1987)
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| Budget Amount *help |
¥27,600,000 (Direct Cost: ¥27,600,000)
Fiscal Year 1987: ¥6,500,000 (Direct Cost: ¥6,500,000)
Fiscal Year 1986: ¥8,600,000 (Direct Cost: ¥8,600,000)
Fiscal Year 1985: ¥12,500,000 (Direct Cost: ¥12,500,000)
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| Keywords | Introduction of DNA and proteins into cells / Lipsomes with gangliosides / Erythrocyte membranes / EF2の構造と機能 / 高分子物質 / 細胞内導入 / 活性ペプチド / 巨大高分子 / 細胞内注入 / 疾病細胞 |
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| Research Abstract |
For studing cellular functions and treatment of genetic and aquired disease cells, efficient introduction of large macromolecules into living cells are important. More than 10 kb DNA can be easily trapped in liposomes by reverse phase evaporation. But, as naked liposomes cannot fuse with normal cells, DNA within liposomes are not introduced into recipient cells. So, after the liposomes incubated with HVJ(Sendai virus) at 37゜C, the mixture was added to cells. DNA was transiently expressed in about 10% of the cells. To increase the efficiency of the introduction, gangliosides were used as one of components of liposomes. When DNA-containing liposomes with gangliosides were added to cells after incubation of the liposomes with HVJ, transient expression of DNA was observed in almost 100% of recipient cells. When erythrocyte membranes were treated with non-ionic detergent in the presence of large macromolecules and then diluted with PBS, the membrane vesicles can enclose such molecules. When
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the vesicles were added to recipient cells after incubation with liposomes with gangliosides and HVJ, macromolecules can be introduced into almost 100% of the cells. When erythrocyte membranes containing proteins and DNA-containing liposomes with gangliosides were mixed and incubated with HVJ and were thenadded to cells, both DNA and proteins were introduced simultaneously into the same cells. These methods are to introduce DNA into the cell cytoplasm, however, as DNA is expressed in the nucleus, DNA should be transported into the nucleus. So, we have studied the mechanism of nuclear transport. Monoclonal antibody against HMG-1 can be transported into the nucleus only when co-introduced into the cells with HMC-1. Binding of HMG-1 to chromatin or DNA was blocked by the antibody. Thus, nuclear protein has two domains for DNA binding and nuclear transport. Possibility for efficient transport of DNA into the nucleus are rised. cDNA of elongation factor 2(EF2) was cloned and sequenced. Amino acids sequence was deduced from the base sequence, and relationship between structure and functions of EF2 was proposed from the results of sequence homology of GTP binding proteins. cDNA of non-ADP ribosylated EF2 was also cloned and amino acid substitution(Gly717-Arg) of the EF2 was found. The cDNA was expressed in transfected cells as toxin resistant. Less
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