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.
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.
Akita
Hidetaka
H
Laboratory for Molecular Design of Pharmaceutics, Graduate School of Pharmaceutical Sciences, Hokkaido University, Sapporo, Hokkaido, Japan. akita@pharm.hokudai.ac.jp
Tanimoto
Mitsuhide
M
Masuda
Tomoya
T
Kogure
Kentaro
K
Hama
Susumu
S
Ninomiya
Keiko
K
Futaki
Shiroh
S
Harashima
Hideyoshi
H
D002467:Cell Nucleus / Q000378:metabolism*; D003593:Cytoplasm / Q000378:metabolism; D003850:Deoxyribonuclease I; D016503:Drug Delivery Systems; D006367:HeLa Cells; D006801:Humans; D008845:Microinjections; D019913:Nuclear Localization Signals; D010957:Plasmids / Q000378:metabolism*; D011107:Polylysine; D012235:Rhodamines
D019913:Nuclear Localization Signals; D012235:Rhodamines; D011107:Polylysine; D003850:Deoxyribonuclease I