Evaluation of the nuclear delivery and intra-nuclear transcription of plasmid DNA condensed with µ (mu) and NLS-µ by cytoplasmic and nuclear microinjection: a comparative study with poly-L-lysine

✍ Scribed by Hidetaka Akita; Mitsuhide Tanimoto; Tomoya Masuda; Kentaro Kogure; Susumu Hama; Keiko Ninomiya; Shiroh Futaki; Hideyoshi Harashima

• Publisher: John Wiley and Sons
• Year: 2006
• Tongue: English
• Weight: 263 KB
• Volume: 8
• Category: Article
• ISSN: 1099-498X
• DOI: 10.1002/jgm.839

✦ Synopsis

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. Copyright © 2005 John Wiley & Sons, Ltd.