Optimization of production and transgene expression of a retrogradely transported pseudotyped lentiviral vector
Lockowandt, Marcus LU ; Günther, Dorothee M. LU ; Quintino, Luis LU
; Breger, Ludvine S.
LU
; Isaksson, Christina
LU
and Lundberg, Cecilia
LU
(2020)
In Journal of Neuroscience Methods
336.
- 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... (More)
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.
(Less)
- author
- Lockowandt, Marcus
LU
; Günther, Dorothee M.
LU
; Quintino, Luis
LU
; Breger, Ludvine S.
LU
; Isaksson, Christina
LU
and Lundberg, Cecilia
LU
- organization
- publishing date
- 2020-04-15
- type
- Contribution to journal
- publication status
- published
- subject
- keywords
- Ago-GFP, FugB2, Rabies virus G-protein, striatum, Substantia nigra
- in
- Journal of Neuroscience Methods
- volume
- 336
- article number
- 108542
- publisher
- Elsevier
- external identifiers
-
- scopus:85080053141
- pmid:32017975
- ISSN
- 0165-0270
- DOI
- 10.1016/j.jneumeth.2019.108542
- language
- English
- LU publication?
- yes
- id
- 6edd0a79-2d70-4d55-8e51-786acd74cdbd
- date added to LUP
- 2020-09-30 13:11:42
- date last changed
- 2024-04-03 15:11:43
@article{6edd0a79-2d70-4d55-8e51-786acd74cdbd,
abstract = {{<p>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.</p>}},
author = {{Lockowandt, Marcus and Günther, Dorothee M. and Quintino, Luis and Breger, Ludvine S. and Isaksson, Christina and Lundberg, Cecilia}},
issn = {{0165-0270}},
keywords = {{Ago-GFP; FugB2; Rabies virus G-protein; striatum; Substantia nigra}},
language = {{eng}},
month = {{04}},
publisher = {{Elsevier}},
series = {{Journal of Neuroscience Methods}},
title = {{Optimization of production and transgene expression of a retrogradely transported pseudotyped lentiviral vector}},
url = {{http://dx.doi.org/10.1016/j.jneumeth.2019.108542}},
doi = {{10.1016/j.jneumeth.2019.108542}},
volume = {{336}},
year = {{2020}},
}