Optimization of production and transgene expression of a retrogradely transported pseudotyped lentiviral vector

• Published in: Journal of neuroscience methods Vol. 336; p. 108542
• Main Authors: Lockowandt, Marcus, Günther, Dorothee M., Quintino, Luis, Breger, Ludvine S., Isaksson, Christina, Lundberg, Cecilia
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 15-04-2020
• Subjects: Ago-GFP; Basic Medicine; FugB2; Medical and Health Sciences; Medicin och hälsovetenskap; Medicinska och farmaceutiska grundvetenskaper; Neurosciences; Neurovetenskaper; Rabies virus G-protein; striatum; Substantia nigra; FugB2; Rabies virus G-protein; Ago-GFP; striatum; Substantia nigra
• ISSN: 0165-0270; 1872-678X
• DOI: 10.1016/j.jneumeth.2019.108542

•Use of polyethylenimine for 293 T transfection improves consistency of viral titers.•The hSyn promoter is more effective in medium spiny neurons compared to CMV.•Lentivirus can be used to transduce a few thousand striatonigral neurons•qRT PCR can be performed by miRNA immunoprecipitation to quantify abundant miRNA.
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.
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.
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.
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.
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.