RRC ID 1985
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)