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 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.
Gourbatsi
Evdoxia
E
Protein Science Group, Department of Biosciences, University of Kent, Canterbury, Kent, CT2 7NJ, UK.
Al-Fageeh
Mohamed B
MB
Marchant
Rosalyn J
RJ
Scott
Sarah J
SJ
Underhill
Michèle F
MF
Smales
C Mark
CM
D000818:Animals; D016466:CHO Cells; D002130:Calcium Phosphates; D002448:Cell Adhesion; D002453:Cell Cycle; D002467:Cell Nucleus / Q000378:metabolism*; D006224:Cricetinae; D003412:Cricetulus; D015870:Gene Expression; D017930:Genes, Reporter / Q000235:genetics; D019913:Nuclear Localization Signals / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D014162:Transfection / Q000379:methods*
D002130:Calcium Phosphates; D019913:Nuclear Localization Signals; C020243:calcium phosphate