Effective gene delivery to adult neurons by a modified form of electroporation.
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.
Leclere
Pascal G
PG
Center for Neuroscience Research, GKT School of Biomedical Sciences, King's College London, Guy's Hospital Campus, London Bridge, London SE1 1UL, United Kingdom.
Panjwani
Aliza
A
Docherty
Reginald
R
Berry
Martin
M
Pizzey
John
J
Tonge
David A
DA
D000367:Age Factors; D000818:Animals; D018929:Cell Culture Techniques / Q000379:methods; D002467:Cell Nucleus / Q000187:drug effects / Q000235:genetics; D002478:Cells, Cultured; D018076:DNA, Complementary / Q000235:genetics / Q000494:pharmacology*; D018274:Electroporation / Q000295:instrumentation / Q000379:methods*; D005727:Ganglia, Spinal / Q000166:cytology / Q000187:drug effects / Q000502:physiology; D005822:Genetic Vectors / Q000235:genetics; D049452:Green Fluorescent Proteins / Q000235:genetics; D018259:Herpesvirus 1, Human / Q000235:genetics; D051381:Rats; D017208:Rats, Wistar; D011930:Reaction Time / Q000187:drug effects / Q000235:genetics; D012165:Retinal Ganglion Cells / Q000166:cytology / Q000187:drug effects / Q000502:physiology*; D013997:Time Factors; D014162:Transfection / Q000295:instrumentation / Q000379:methods*
D018076:DNA, Complementary; D049452:Green Fluorescent Proteins