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

Kim, J.-H., Dittgen, T., Nimmerjahn, A., Waters, D. J., Pawlak, V., Helmchen, F., et al. (2004). Sindbis vector SINrep(nsP2S726): a tool for rapid heterologous expression with attenuated cytotoxicity in neurons. Journal of Neuroscience Methods, 133(1), 81-90. doi:10.1016/j.jneumeth.2003.09.029.

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Item Permalink: http://hdl.handle.net/11858/00-001M-0000-002A-2B24-4 Version Permalink: http://hdl.handle.net/11858/00-001M-0000-002A-2B25-2
Genre: Journal Article
Alternative Title : Sindbis vector SINrep(nsP2S726): a tool for rapid heterologous expression with attenuated cytotoxicity in neurons

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 Creators:
Kim, Jin-Hyun, Author
Dittgen, Tanjew1, Author              
Nimmerjahn, Axel2, Author              
Waters, David Jack2, Author              
Pawlak, Verena1, 2, Author              
Helmchen, Fritjof2, Author              
Schlesinger, Sondra, Author
Seeburg, Peter H.1, Author              
Osten, Pavel1, Author              
Affiliations:
1Department of Molecular Neurobiology, Max Planck Institute for Medical Research, Max Planck Society, ou_1497704              
2Department of Cell Physiology, Max Planck Institute for Medical Research, Max Planck Society, ou_1497701              

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Free keywords: Sindbis virus; Heterologous protein expression; Neuron; In vitro; In vivo
 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.

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Language(s): eng - English
 Dates: 2003-09-232003-08-132003-09-252003-12-232004-02-15
 Publication Status: Published in print
 Pages: 9
 Publishing info: -
 Table of Contents: -
 Rev. Method: Peer
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Title: Journal of Neuroscience Methods
  Other : J. Neurosci. Meth.
Source Genre: Journal
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Publ. Info: Amsterdam : Elsevier
Pages: - Volume / Issue: 133 (1) Sequence Number: - Start / End Page: 81 - 90 Identifier: ISSN: 0165-0270
CoNE: https://pure.mpg.de/cone/journals/resource/954925480594