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High-efficiency gene transfer into cultured embryonic motoneurons using recombinant lentiviruses

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Abstract

Primary neurons are a common tool for investigating gene function for survival and morphological and functional differentiation. Gene transfer techniques play an important role in this context. However, the efficacy of conventional gene transfer techniques, in particular for primary motoneurons is low so that it is not possible to distinguish whether the observed effects are representative for all neurons or only for the small subpopulation that expresses the transfected cDNA. In order to develop techniques that allow high gene transfer rates, we have optimized lentiviral-based gene transfer for cultured motoneurons by using a replication-defective viral vector system. These techniques result in transduction efficacies higher than 50%, as judged by EGFP expression under the control of SFFV or CMV promoters. Under the same conditions, survival and morphology of the cultured motoneurons was not altered, at least not when virus titers did not exceed a multiplicity of infection of 100. Under the same cell culture conditions, electroporation resulted in less than 5% transfected motoneurons and reduced survival. Therefore we consider this lentivirus-based gene transfer protocol as a suitable tool to study the effects of gene transfer on motoneuron survival, differentiation and function.

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Acknowledgments

We thank Martin Heinkelein for providing the initial viral plasmids and support in establishing lentiviral methods as well as Katrin Kuebert for technical assistance. This work was supported by the Deutsche Forschungsgemeinschaft (SFB581), the IKFZ, the Hermann und Lilly Schilling-Stiftung, the SMA Foundation, and the EU through the APOPIS project.

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Authors and Affiliations

  1. Institute for Clinical Neurobiology, University of Wurzburg, Josef-Schneider-Str. 11, 97080, Wurzburg, Germany

    Florian L. P. Bender, Matthias Fischer, Natalja Funk, Nadiya Orel & Michael Sendtner

  2. Institute for Virology and Immunobiology, University of Wurzburg, Versbacher Str. 7, 97078, Wurzburg, Germany

    Axel Rethwilm

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  1. Florian L. P. Bender
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  2. Matthias Fischer
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  3. Natalja Funk
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  4. Nadiya Orel
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  5. Axel Rethwilm
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  6. Michael Sendtner
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Correspondence to Michael Sendtner.

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Bender, F.L.P., Fischer, M., Funk, N. et al. High-efficiency gene transfer into cultured embryonic motoneurons using recombinant lentiviruses. Histochem Cell Biol 127, 439–448 (2007). https://doi.org/10.1007/s00418-006-0247-5

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