Sindbis vector SINrep(nsP2S726): a tool for rapid heterologous expression with attenuated cytotoxicity in neurons

J Neurosci Methods. 2004 Feb 15;133(1-2):81-90. doi: 10.1016/j.jneumeth.2003.09.029.


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.

Publication types

  • Comparative Study
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Autoradiography / methods
  • Blotting, Northern / methods
  • Blotting, Western / methods
  • Cell Survival
  • Cells, Cultured
  • Cricetinae
  • Cysteine Endopeptidases / metabolism
  • Embryo, Mammalian
  • Gene Expression Regulation, Viral / physiology*
  • Gene Transfer Techniques
  • Genetic Vectors / genetics
  • Green Fluorescent Proteins
  • Hippocampus / cytology
  • Luminescent Proteins / metabolism
  • Membrane Potentials / physiology
  • Mutation
  • Neural Conduction / physiology
  • Neurons / metabolism*
  • Neurons / virology
  • Patch-Clamp Techniques
  • Phosphopyruvate Hydratase
  • Rats
  • Sindbis Virus / genetics*
  • Time Factors


  • Luminescent Proteins
  • Green Fluorescent Proteins
  • Cysteine Endopeptidases
  • nsP2 proteinase
  • Phosphopyruvate Hydratase