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

2003; Elsevier BV; Volume: 133; Issue: 1-2 Linguagem: Inglês

10.1016/j.jneumeth.2003.09.029

1872-678X

Jinhyun Kim, Tanjew Dittgen, Axel Nimmerjahn, Jack Waters, Verena Pawlak, Fritjof Helmchen, Sondra Schlesinger, Peter H. Seeburg, Pavel Osten,

interferon and immune responses

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(nsP2S726) 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(nsP2S726) 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(nsP2S726) 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.