Effective gene delivery to adult neurons by a modified form of electroporation

• Published in: Journal of neuroscience methods Vol. 142; no. 1; pp. 137 - 143
• Main Authors: Leclere, Pascal G., Panjwani, Aliza, Docherty, Reginald, Berry, Martin, Pizzey, John, Tonge, David A.
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 15.03.2005
• Subjects: Age Factors; Animals; Cell Culture Techniques - methods; Cell Nucleus - drug effects; Cell Nucleus - genetics; Cells, Cultured; DNA, Complementary - genetics; DNA, Complementary - pharmacology; DRG; Electroporation - instrumentation; Electroporation - methods; Ganglia, Spinal - cytology; Ganglia, Spinal - drug effects; Ganglia, Spinal - physiology; Gene transfer; Genetic Vectors - genetics; GFP; Green Fluorescent Proteins - genetics; Herpesvirus 1, Human - genetics; HSV-1; Nucleofection; Rats; Rats, Wistar; Reaction Time - drug effects; Reaction Time - genetics; Retinal Ganglion Cells - cytology; Retinal Ganglion Cells - drug effects; Retinal Ganglion Cells - physiology; Retinal ganglion neurons; Time Factors; Transfection; Transfection - instrumentation; Transfection - methods; GFP; HSV-1; Transfection; Nucleofection; Gene transfer; DRG; Retinal ganglion neurons
• ISSN: 0165-0270; 1872-678X
• DOI: 10.1016/j.jneumeth.2004.08.012

Non-viral methods of transfection of cDNAs into adult neurons and other post-mitotic cells are generally very inefficient. However, the recent development of Nucleofector™ technology developed by Amaxa Biosystems allows direct delivery of cDNAs into the nucleus, enabling transfection of non-dividing cells. In this study, we describe a reliable method for culturing large numbers of retinal cells from adult rats and using Nucleofection, we were able to transfect cDNA-encoding GFP (jellyfish green fluorescent protein) into retinal ganglion cells (RGCs) with relatively high efficiency (up to 28%). Neuronal GFP expression was observed within 18 h and continued for up to 14 days. This compares with values up to 60% of RGCs expressing GFP following infection with an HSV-1 vector. Adult rat dorsal root ganglion (DRG) neurons were also successfully transfected. Thus, in summary, Nucleofection provides the possibility for a fast and efficient method for cDNA delivery and study of gene function in adult mammalian neurons.