論文 - 詳細
RRC ID | 1985 |
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著者 | Akita H, Tanimoto M, Masuda T, Kogure K, Hama S, Ninomiya K, Futaki S, Harashima H. |
タイトル | Evaluation of the nuclear delivery and intra-nuclear transcription of plasmid DNA condensed with micro (mu) and NLS-micro by cytoplasmic and nuclear microinjection: a comparative study with poly-L-lysine. |
ジャーナル | J Gene Med |
Abstract |
BACKGROUND:The efficient nuclear delivery of plasmid DNA (pDNA) is essential for the development of a promising non-viral gene vector. In an attempt to achieve nuclear delivery, NLS-mu, a novel pDNA condenser, was prepared. This consists of mu, a highly potent polypeptide for condensing the pDNA, and a SV40 T antigen-derived nuclear localization signal (NLS(SV40)). METHODS:The utility of NLS-mu was assessed in terms of green fluorescent protein (GFP) expression after cytoplasmic and nuclear microinjection of GFP-encoding pDNA along with the transfection, and compared with mu and poly-L-lysine (PLL). Trans-gene expression after cytoplasmic microinjection was affected by the efficiencies of nuclear transfer and following intra-nuclear transcription. To evaluate the nuclear transfer process separately, we introduced a parameter, a nuclear transfer score (NT score), which was calculated as the trans-gene expression after cytoplasmic microinjection divided by that after nuclear microinjection. RESULTS:As expected, the rank order of trans-gene expression after the transfection and cytoplasmic microinjection was NLS-mu > mu > PLL. However, the calculated NT scores were unexpectedly ranked as mu = NLS-mu > PLL, suggesting that mu, and not NLS(SV40), is responsible for the nuclear delivery of pDNA. In addition, confocal images of rhodamine-labeled pDNA indicated that pDNA condensed with mu and NLS-mu was delivered as a condensed form. In comparing the nuclear transcription, the rank order of trans-gene expression after nuclear microinjection was PLL = NLS-mu > mu, suggesting that intra-nuclear transcription is inhibited by efficient condensation by mu, and is avoided by the attachment of NLS(SV40). CONCLUSIONS:Collectively, NLS-mu, which consists of chimeric functions, is an excellent DNA condenser, and the process is based on mu-derived nuclear transfer and NLS(SV40)-derived efficient intra-nuclear transcription. |
巻・号 | 8(2) |
ページ | 198-206 |
公開日 | 2006-2-1 |
DOI | 10.1002/jgm.839 |
PMID | 16285003 |
MeSH | Cell Nucleus / metabolism* Cytoplasm / metabolism Deoxyribonuclease I Drug Delivery Systems* HeLa Cells Humans Microinjections Nuclear Localization Signals* Plasmids / metabolism* Polylysine* Rhodamines |
IF | 3.258 |
引用数 | 29 |
WOS 分野 | MEDICINE, RESEARCH & EXPERIMENTAL BIOTECHNOLOGY & APPLIED MICROBIOLOGY GENETICS & HEREDITY |
リソース情報 | |
ヒト・動物細胞 | HeLa(RCB0007) |