Overexpression of proteins in neurons using replication-deficient virus.
Overexpression of proteins is a powerful way to determine their function. Until recently, the low efficiency of neuronal transfection has made it difficult to use overexpression and structure-function studies to investigate the role of neuronal proteins in their native environment. The development of neurotrophic viral systems has overcome the obstacle of low efficiency and allows for unprecedented opportunities to use biochemical and electrophysiological techniques to assess the effects of overexpressing wild-type or mutant proteins in neurons. Here, a general protocol for the production of replication-deficient Semliki Forest virus constructs directing the overexpression of proteins of interest in cultured mammalian neurons is described.
Ahlquist
Richard M
RM
Department of Physiology and Biophysics, University of Washington, Seattle, USA.
Sullivan
Jane M
JM
D000818:Animals; D002478:Cells, Cultured; D003001:Cloning, Molecular; D006224:Cricetinae; D004247:DNA / Q000235:genetics; D018076:DNA, Complementary / Q000235:genetics; D003673:Defective Viruses / Q000235:genetics* / Q000502:physiology*; D015870:Gene Expression; D009474:Neurons / Q000378:metabolism*; D010957:Plasmids / Q000235:genetics; D016133:Polymerase Chain Reaction; D011506:Proteins / Q000235:genetics*; D012672:Semliki forest virus / Q000502:physiology; D014158:Transcription, Genetic; D014771:Virion; D014775:Virus Activation; D014779:Virus Replication
D018076:DNA, Complementary; D011506:Proteins; D004247:DNA