Sindbis vector SINrep(nsP2S726): a tool for rapid heterologous expression with attenuated cytotoxicity in neurons.
Sindbis virus-based vectors have been successfully used for transient heterologous protein expression in neurons. Their main limitation arises from infection-associated cytotoxicity, attributed largely to a progressive shut down of host cell protein synthesis. Here we evaluated a modified Sindbis vector, based on a viral strain containing a point mutation in the second nonstructural protein, nsP2 P726S, described to delay inhibition of protein synthesis in BHK cells [Virology 228 (1997) 74], for heterologous expression in neurons in vitro and in vivo. First, we constructed an optimized helper vector, termed DH-BB(tRNA/TE12), for production of SINrep(nsP2S(726)) viral particles with low levels of helper RNA co-packaging and high neurospecificity of infection. Second, we determined that hippocampal primary neurons infected with SINrep(nsP2S(726)) virus expressing EGFP showed a delayed onset of viral induced cytotoxicity and higher levels of EGFP expression in comparison to cells infected with wild type SINrep5 EGFP-expressing virus. However, a strong decrease in protein synthesis still occurred by day 3 postinfection. The SINrep(nsP2S(726)) vector is thus well suited for rapid high level expression within this time window. As an experimental example, we demonstrate the applicability of this system for high-resolution two-photon imaging of dendritic spines in vivo.
Kim
Jinhyun
J
Max-Planck Institute for Medical Research, Department of Molecular Neurobiology, Jahnstrasse 29, 69120 Heidelberg, Germany.
Dittgen
Tanjew
T
Nimmerjahn
Axel
A
Waters
Jack
J
Pawlak
Verena
V
Helmchen
Fritjof
F
Schlesinger
Sondra
S
Seeburg
Peter H
PH
Osten
Pavel
P
D000818:Animals; D001345:Autoradiography / Q000379:methods; D015152:Blotting, Northern / Q000379:methods; D015153:Blotting, Western / Q000379:methods; D002470:Cell Survival; D002478:Cells, Cultured; D006224:Cricetinae; D003546:Cysteine Endopeptidases / Q000378:metabolism; D004622:Embryo, Mammalian; D015967:Gene Expression Regulation, Viral / Q000502:physiology*; D018014:Gene Transfer Techniques; D005822:Genetic Vectors / Q000235:genetics; D049452:Green Fluorescent Proteins; D006624:Hippocampus / Q000166:cytology; D008164:Luminescent Proteins / Q000378:metabolism; D008564:Membrane Potentials / Q000502:physiology; D009154:Mutation; D009431:Neural Conduction / Q000502:physiology; D009474:Neurons / Q000378:metabolism* / Q000821:virology; D018408:Patch-Clamp Techniques; D010751:Phosphopyruvate Hydratase; D051381:Rats; D012845:Sindbis Virus / Q000235:genetics*; D013997:Time Factors
D008164:Luminescent Proteins; D049452:Green Fluorescent Proteins; D003546:Cysteine Endopeptidases; C078306:nsP2 proteinase; D010751:Phosphopyruvate Hydratase