Published online 11 July 2006
© 2006 The Author(s)
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Methods Online |
Cytoplasmic expression systems triggered by mRNA yield increased gene expression in post-mitotic neurons
Department of Clinical Pharmacology, University of Oxford Oxford OX2 6HE, UK 1 Department of Anesthesia and Critical Care, Massachusetts General Hospital and Harvard Medical School Boston, MA 02129, USA 2 Department of Biochemistry and Molecular Biology, University of British Columbia Vancouver, British Columbia, Canada 3 Molecular Neuroscience Group, Department of Medicine, University of Birmingham Birmingham B15 2TT, UK
*To whom correspondence should be addressed. Tel: +44 1865 224986; Fax: +44 1865 224538; Email: Len.Seymour{at}clinpharm.ox.ac.uk
Received February 23, 2006. Revised May 26, 2006. Accepted June 7, 2006.
Non-viral vectors are promising vehicles for gene therapy but delivery of plasmid DNA to post-mitotic cells is challenging as nuclear entry is particularly inefficient. We have developed and evaluated a hybrid mRNA/DNA system designed to bypass the nuclear barrier to transfection and facilitate cytoplasmic gene expression. This system, based on co-delivery of mRNA(A64) encoding for T7 RNA polymerase (T7 RNAP) with a T7-driven plasmid, produced between 10- and 2200-fold higher gene expression in primary dorsal root ganglion neuronal (DRGN) cultures isolated from Sprague–Dawley rats compared to a cytomegalovirus (CMV)-driven plasmid, and 30-fold greater expression than the enhanced T7-based autogene plasmid pR011. Cell-free assays and in vitro transfections highlighted the versatility of this system with small quantities of T7 RNAP mRNA required to mediate expression at levels that were significantly greater than with the T7-driven plasmid alone or supplemented with T7 RNAP protein. We have also characterized a number of parameters, such as mRNA structure, intracellular stability and persistence of each nucleic acid component that represent important factors in determining the transfection efficiency of this hybrid expression system. The results from this study demonstrate that co-delivery of mRNA is a promising strategy to yield increased expression with plasmid DNA, and represents an important step towards improving the capability of non-viral vectors to mediate efficient gene transfer in cell types, such as in DRGN, where the nuclear membrane is a significant barrier to transfection.