High-efficiency gene transfer into cultured embryonic motoneurons using recombinant lentiviruses.
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.
Bender
Florian L P
FL
Institute for Clinical Neurobiology, University of Wurzburg, Josef-Schneider-Str. 11, 97080, Wurzburg, Germany.
Fischer
Matthias
M
Funk
Natalja
N
Orel
Nadiya
N
Rethwilm
Axel
A
Sendtner
Michael
M
D000818:Animals; D002460:Cell Line; D002478:Cells, Cultured; D003587:Cytomegalovirus / Q000235:genetics; D003673:Defective Viruses / Q000235:genetics; D018274:Electroporation / Q000379:methods; D005822:Genetic Vectors / Q000235:genetics; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D007150:Immunohistochemistry; D016086:Lentivirus / Q000235:genetics*; D051379:Mice; D008810:Mice, Inbred C57BL; D018613:Microscopy, Confocal; D009046:Motor Neurons / Q000166:cytology / Q000378:metabolism*; D011401:Promoter Regions, Genetic / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D013155:Spleen Focus-Forming Viruses / Q000235:genetics; D013997:Time Factors; D014162:Transfection / Q000379:methods*
D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins