Neuronal expression and cell-type-specific gene-silencing of best1 in thalamic reticular nucleus neurons using psico-red system

Jae Young Jung, Seung Eun Lee, Eun Mi Hwang, Changjoon Lee

Research output: Contribution to journalArticle

3 Citations (Scopus)

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 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.

Original languageEnglish
Pages (from-to)120-129
Number of pages10
JournalExperimental Neurobiology
Volume25
Issue number3
DOIs
Publication statusPublished - 2016 Jun 1

Fingerprint

Thalamic Nuclei
Gene Silencing
Neurons
Homeobox Genes
Fluorescence
Small Interfering RNA
Genes
Viruses
Parvalbumins
Proteins
Reporter Genes
Transgenic Mice
Immunohistochemistry

Keywords

  • Bestrophin-1
  • DLX-cre
  • Parvalbumin
  • PSico
  • Thalamic reticular nucleus
  • Virus

ASJC Scopus subject areas

  • Clinical Neurology
  • Cellular and Molecular Neuroscience

Cite this

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, Changjoon.

In: Experimental Neurobiology, Vol. 25, No. 3, 01.06.2016, p. 120-129.

Research output: Contribution to journalArticle

Jung, Jae Young ; Lee, Seung Eun ; Hwang, Eun Mi ; Lee, Changjoon. / Neuronal expression and cell-type-specific gene-silencing of best1 in thalamic reticular nucleus neurons using psico-red system. In: Experimental Neurobiology. 2016 ; Vol. 25, No. 3. pp. 120-129.
@article{bb5aea846fd54b13a5e5b44398822326,
title = "Neuronal expression and cell-type-specific gene-silencing of best1 in thalamic reticular nucleus neurons using psico-red system",
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 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.",
keywords = "Bestrophin-1, DLX-cre, Parvalbumin, PSico, Thalamic reticular nucleus, Virus",
author = "Jung, {Jae Young} and Lee, {Seung Eun} and Hwang, {Eun Mi} and Changjoon Lee",
year = "2016",
month = "6",
day = "1",
doi = "10.5607/en.2016.25.3.120",
language = "English",
volume = "25",
pages = "120--129",
journal = "Experimental Neurobiology",
issn = "1226-2560",
publisher = "Korean Society for Brain and Neural Science",
number = "3",

}

TY - JOUR

T1 - Neuronal expression and cell-type-specific gene-silencing of best1 in thalamic reticular nucleus neurons using psico-red system

AU - Jung, Jae Young

AU - Lee, Seung Eun

AU - Hwang, Eun Mi

AU - Lee, Changjoon

PY - 2016/6/1

Y1 - 2016/6/1

N2 - 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 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.

AB - 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 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.

KW - Bestrophin-1

KW - DLX-cre

KW - Parvalbumin

KW - PSico

KW - Thalamic reticular nucleus

KW - Virus

UR - http://www.scopus.com/inward/record.url?scp=84995678211&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=84995678211&partnerID=8YFLogxK

U2 - 10.5607/en.2016.25.3.120

DO - 10.5607/en.2016.25.3.120

M3 - Article

AN - SCOPUS:84995678211

VL - 25

SP - 120

EP - 129

JO - Experimental Neurobiology

JF - Experimental Neurobiology

SN - 1226-2560

IS - 3

ER -