Marcus Lockowandt1, Dorothee M Günther1, Luis Quintino1, Ludvine S Breger1, Christina Isaksson1, Cecilia Lundberg2. 1. CNS Gene therapy Unit, Wallenberg Neuroscience Center, Dept Experimental Sciences, Lund University BMC A11, 22184 Lund, Sweden. 2. CNS Gene therapy Unit, Wallenberg Neuroscience Center, Dept Experimental Sciences, Lund University BMC A11, 22184 Lund, Sweden. Electronic address: Cecilia.Lundberg@med.lu.se.
Abstract
BACKGROUND: To target specific neuronal populations by gene transfer is challenging. A complicating fact is that populations of neurons may have opposing roles despite being found adjacent to each other. One example is the medium spiny neurons of the striatum. These cells have different projection patterns, a trait used in this study to specifically target one population. NEW METHOD: Here we present a way of labeling and further studying neurons based on their projections. This was achieved by pseudotyping lentiviral vectors with a chimeric glycoprotein allowing for retrograde transport in combination with optimizing the promoter element used. RESULTS: We transduced on average 4000 neurons of the direct pathway in the striatum, with the viral vector allowing for microscopy and miRNA immunoprecipitation. In addition, we were able to optimize vector production, reducing the time and material used. COMPARISON WITH EXISTING METHOD: The optimized protocol is more reproducible compared to previously published protocols. Alternative methods to study specific populations of neurons are transgenic animals or, if available, specific promoter elements. However, very specific promoter elements are rarely available and often large, limiting the usefulness in viral vectors. Our optimized retrograde vectors allow for selection based on neuronal projections and are therefore independent of such elements. CONCLUSION: We have developed a method that allows for specific analysis of neuronal subpopulations in the brain either by microscopy or by biochemical methods e.g. immunoprecipitation. This method is simple to use and can be combined with transgenic animals for studying disease models.
BACKGROUND: To target specific neuronal populations by gene transfer is challenging. A complicating fact is that populations of neurons may have opposing roles despite being found adjacent to each other. One example is the medium spiny neurons of the striatum. These cells have different projection patterns, a trait used in this study to specifically target one population. NEW METHOD: Here we present a way of labeling and further studying neurons based on their projections. This was achieved by pseudotyping lentiviral vectors with a chimeric glycoprotein allowing for retrograde transport in combination with optimizing the promoter element used. RESULTS: We transduced on average 4000 neurons of the direct pathway in the striatum, with the viral vector allowing for microscopy and miRNA immunoprecipitation. In addition, we were able to optimize vector production, reducing the time and material used. COMPARISON WITH EXISTING METHOD: The optimized protocol is more reproducible compared to previously published protocols. Alternative methods to study specific populations of neurons are transgenic animals or, if available, specific promoter elements. However, very specific promoter elements are rarely available and often large, limiting the usefulness in viral vectors. Our optimized retrograde vectors allow for selection based on neuronal projections and are therefore independent of such elements. CONCLUSION: We have developed a method that allows for specific analysis of neuronal subpopulations in the brain either by microscopy or by biochemical methods e.g. immunoprecipitation. This method is simple to use and can be combined with transgenic animals for studying disease models.
Authors: Marcus Davidsson; Matilde Negrini; Swantje Hauser; Alexander Svanbergsson; Marcus Lockowandt; Giuseppe Tomasello; Fredric P Manfredsson; Andreas Heuer Journal: Sci Rep Date: 2020-12-09 Impact factor: 4.379
Authors: Eliška Waloschková; Ana Gonzalez-Ramos; Apostolos Mikroulis; Jan Kudláček; My Andersson; Marco Ledri; Merab Kokaia Journal: Int J Mol Sci Date: 2021-12-08 Impact factor: 5.923