A modified Sindbis vector for prolonged gene expression in neurons.
Sindbis viruses have been widely used in neurobiology to express a variety of genes in cultured neurons, in cultured slices, and in vivo. They provide fast onset and high levels of expression of foreign genes, but the expression is limited to a short time window due to a shut-off of host protein synthesis. We have used a mutation in an essential gene (nsP2) of the life cycle of Sindbis, which allows the functional analysis of changes in protein expression for >/=6 days after infection. This Sindbis mutant (nsP2) was used to express enhanced green fluorescent protein (EGFP) in hippocampal neurons in culture and in vivo without any sign of toxicity, based on two-photon imaging and electrophysiology. In addition, the EGFP mutant virus can be injected in vivo to visualize spines and other details of neuronal structure. The Sindbis mutant described here provides an improved tool in neurobiology with reduced cytotoxicity and a prolonged time window of expression for novel applications in imaging and behavior. In addition, the use of this vector for the functional expression of mammalian voltage-gated ion channels in organotypic slices is demonstrated.
Jeromin
Andreas
A
Division of Neuroscience, Baylor College of Medicine, Houston, Texas 77030, USA. jeromin@ltp.neusc.bcm.tmc.edu
Yuan
Li-Lian
LL
Frick
Andreas
A
Pfaffinger
Paul
P
Johnston
Daniel
D
D000818:Animals; D000831:Animals, Newborn; D015967:Gene Expression Regulation, Viral / Q000502:physiology*; D005822:Genetic Vectors / Q000096:biosynthesis* / Q000235:genetics; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins / Q000096:biosynthesis / Q000235:genetics; D009474:Neurons / Q000378:metabolism*; D009924:Organ Culture Techniques; D051381:Rats; D017207:Rats, Sprague-Dawley; D012845:Sindbis Virus / Q000235:genetics* / Q000378:metabolism*
D008164:Luminescent Proteins; D049452:Green Fluorescent Proteins