Cargando…

Neuronal Expression and Cell-Type-Specific Gene-Silencing of Best1 in Thalamic Reticular Nucleus Neurons Using pSico-Red System

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...

Descripción completa

Detalles Bibliográficos
Autores principales: Jung, Jae-Young, Lee, Seung Eun, Hwang, Eun Mi, Lee, C. Justin
Formato: Online Artículo Texto
Lenguaje:English
Publicado: The Korean Society for Brain and Neural Science 2016
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4923356/
https://www.ncbi.nlm.nih.gov/pubmed/27358580
http://dx.doi.org/10.5607/en.2016.25.3.120
_version_ 1782439716141924352
author Jung, Jae-Young
Lee, Seung Eun
Hwang, Eun Mi
Lee, C. Justin
author_facet Jung, Jae-Young
Lee, Seung Eun
Hwang, Eun Mi
Lee, C. Justin
author_sort Jung, Jae-Young
collection PubMed
description 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 adeno-associated 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 Best1-shRNA. 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.
format Online
Article
Text
id pubmed-4923356
institution National Center for Biotechnology Information
language English
publishDate 2016
publisher The Korean Society for Brain and Neural Science
record_format MEDLINE/PubMed
spelling pubmed-49233562016-06-29 Neuronal Expression and Cell-Type-Specific Gene-Silencing of Best1 in Thalamic Reticular Nucleus Neurons Using pSico-Red System Jung, Jae-Young Lee, Seung Eun Hwang, Eun Mi Lee, C. Justin Exp Neurobiol Original Article 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 adeno-associated 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 Best1-shRNA. 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. The Korean Society for Brain and Neural Science 2016-06 2016-06-22 /pmc/articles/PMC4923356/ /pubmed/27358580 http://dx.doi.org/10.5607/en.2016.25.3.120 Text en Copyright © Experimental Neurobiology 2016. http://creativecommons.org/licenses/by-nc/4.0 This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
spellingShingle Original Article
Jung, Jae-Young
Lee, Seung Eun
Hwang, Eun Mi
Lee, C. Justin
Neuronal Expression and Cell-Type-Specific Gene-Silencing of Best1 in Thalamic Reticular Nucleus Neurons Using pSico-Red System
title Neuronal Expression and Cell-Type-Specific Gene-Silencing of Best1 in Thalamic Reticular Nucleus Neurons Using pSico-Red System
title_full Neuronal Expression and Cell-Type-Specific Gene-Silencing of Best1 in Thalamic Reticular Nucleus Neurons Using pSico-Red System
title_fullStr Neuronal Expression and Cell-Type-Specific Gene-Silencing of Best1 in Thalamic Reticular Nucleus Neurons Using pSico-Red System
title_full_unstemmed Neuronal Expression and Cell-Type-Specific Gene-Silencing of Best1 in Thalamic Reticular Nucleus Neurons Using pSico-Red System
title_short Neuronal Expression and Cell-Type-Specific Gene-Silencing of Best1 in Thalamic Reticular Nucleus Neurons Using pSico-Red System
title_sort neuronal expression and cell-type-specific gene-silencing of best1 in thalamic reticular nucleus neurons using psico-red system
topic Original Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4923356/
https://www.ncbi.nlm.nih.gov/pubmed/27358580
http://dx.doi.org/10.5607/en.2016.25.3.120
work_keys_str_mv AT jungjaeyoung neuronalexpressionandcelltypespecificgenesilencingofbest1inthalamicreticularnucleusneuronsusingpsicoredsystem
AT leeseungeun neuronalexpressionandcelltypespecificgenesilencingofbest1inthalamicreticularnucleusneuronsusingpsicoredsystem
AT hwangeunmi neuronalexpressionandcelltypespecificgenesilencingofbest1inthalamicreticularnucleusneuronsusingpsicoredsystem
AT leecjustin neuronalexpressionandcelltypespecificgenesilencingofbest1inthalamicreticularnucleusneuronsusingpsicoredsystem