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

Kim, J., Dittgen, T., Nimmerjahn, A., Waters, J., Pawlak, V., Helmchen, F., Schlesinger, S., Seeburg, P. H., Osten, P. (February 2004) Sindbis vector SINrep(nsP2S726): a tool for rapid heterologous expression with attenuated cytotoxicity in neurons. Journal of Neuroscience Methods, 133 (1-2). pp. 81-90. ISSN 0165-0270

URL: http://www.ncbi.nlm.nih.gov/pubmed/14757348
DOI: 10.1016/j.jneumeth.2003.09.029

Abstract

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.

Item Type: Paper
Additional Information:
Uncontrolled Keywords: 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 virology Patch-Clamp Techniques Phosphopyruvate Hydratase Rats Sindbis Virus genetics Time Factors
Subjects: therapies > viral vectors
CSHL Authors:
Communities: CSHL labs > Osten lab
Depositing User: CSHL Librarian
Date: 15 February 2004
Date Deposited: 01 Feb 2012 16:53
Last Modified: 08 Apr 2013 15:42
Related URLs:
URI: http://repository.cshl.edu/id/eprint/22412

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