Noncovalently linked nuclear localization peptides for enhanced calcium phosphate transfection

The generation of cell lines stably expressing recombinant material is a lengthy process and there has thus been much interest in the use of transient expression systems to rapidly produce recombinant material. To achieve this, the DNA of interest must be delivered into the nucleus of the target cel...

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Bibliographic Details
Published in:Molecular biotechnology 2006-05, Vol.33 (1), p.1-11
Main Authors: Gourbatsi, Evdoxia, Al-Fageeh, Mohamed B, Marchant, Rosalyn J, Scott, Sarah J, Underhill, Michèle F, Smales, C Mark
Format: Article
Language:English
Subjects:
Animals
Calcium Phosphates
Cell Adhesion
Cell Cycle
Cell Nucleus - metabolism
CHO Cells
Cricetinae
Cricetulus
Deoxyribonucleic acid
DNA
Gene Expression
Genes, Reporter - genetics
Nuclear Localization Signals - chemistry
Nuclear Localization Signals - genetics
Nuclear Localization Signals - metabolism
Peptides
Proteins
Simian virus 40
Studies
Transfection - methods
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Summary:The generation of cell lines stably expressing recombinant material is a lengthy process and there has thus been much interest in the use of transient expression systems to rapidly produce recombinant material. To achieve this, the DNA of interest must be delivered into the nucleus of the target cell. The mechanisms by which this process occurs are poorly understood and the efficiency of various methods differs widely. Recently, nuclear localization signals (NLSs) have been investigated to target entry of DNA into the nucleus of mammalian cells. We have used NLSs from the SV40 and Tat antigens mixed with our model luciferase reporter gene plasmid for the transfection of Chinese hamster ovary (CHO) cells using calcium phosphate and FuGENE 6 transfection technology. The noncovalent complexation of NLSs with plasmid DNA before calcium phosphate-mediated transfection resulted in enhanced reporter gene expression with increasing ratios of NLS to plasmid until reaching a maximum. At higher ratios than maximum expression, the expression levels decreased. On the other hand, when using FuGENE 6 reagent NLSs did not enhance reporter gene expression. Cell cycle arrest in G(2)/M phase obliterated the effect of the NLS on reporter gene expression when using the calcium phosphate transfection method.
ISSN:1073-6085
1559-0305
DOI:10.1385/MB:33:1:1