Neuronal Expression and Cell-Type-Specific Gene-Silencing of Best1 in Thalamic Reticular Nucleus Neurons Using pSico-Red System
- Authors
- Jung, Jae-Young; Lee, Seung Eun; Hwang, Eun Mi; Lee, C. Justin
- Issue Date
- 6월-2016
- Publisher
- KOREAN SOC BRAIN & NEURAL SCIENCE, KOREAN SOC NEURODEGENERATIVE DISEASE
- Keywords
- bestrophin-1; thalamic reticular nucleus; pSico; virus; DLX-cre; parvalbumin
- Citation
- EXPERIMENTAL NEUROBIOLOGY, v.25, no.3, pp.120 - 129
- Indexed
- SCOPUS
KCI
- Journal Title
- EXPERIMENTAL NEUROBIOLOGY
- Volume
- 25
- Number
- 3
- Start Page
- 120
- End Page
- 129
- URI
- https://scholar.korea.ac.kr/handle/2021.sw.korea/88458
- DOI
- 10.5607/en.2016.25.3.120
- ISSN
- 1226-2560
- Abstract
- Assessing the cell-type expression pattern of a certain gene can be achieved by using cell-type-specific gene manipulation. Recently, cre-recombinase-dependent gene-silencing tool, pSico has become popular in neuroscientific research. However, pSico has a critical limitation that gene-silenced cell cannot be identified by fluorescence, due to an excision of the reporter gene for green fluorescence protein (GFP). To overcome this limitation, we newly developed pSico-Red, with mCherry gene as a reporter outside two loxP sites, so that red mCherry signal is detected in all transfected cells. When a cell expresses cre, GFP is excised and shRNA is enabled, resulting in disappearance of GFP. This feature of pSico-Red provides not only cell-type-specific gene-silencing but also identification of cre expressing cells. Using this system, we demonstrated for the first time the neuronal expression of the Bestrophin-1 (Best1) in thalamic reticular nucleus (TRN) and TRN-neuron-specific gene-silencing of Best1. We combined adenoassociated virus (AAV) carrying Best1-shRNA in pSico-Red vector and transgenic mouse expressing cre under the promoter of distal-less homeobox 5/6 (DLX5/6), a marker for inhibitory neurons. Firstly, we found that almost all of inhibitory neurons in TRN express Best1 by immunohistochemistry. Using pSico-Red virus, we found that 80% of infected TRN neurons were DLX5/6-cre positive but parvalbumin negative. Finally, we found that Best1 in DLX5/6-cre positive neurons were significantly reduced by Best1shRNA. Our study demonstrates that TRN neurons strongly express Best1 and that pSico-Red is a valuable tool for cell-type-specific gene manipulation and identification of specific cell population.
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Collections - Graduate School > KU-KIST Graduate School of Converging Science and Technology > 1. Journal Articles
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