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 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.
authors.affiliation: Center for Neuroscience and Functional Connectomics, Brain Science Institute, Korea Institute of Science and Technology (KIST), Seoul 02792, Korea.; Neuroscience Program, University of Science and Technology (UST), Daejeon 34113, Korea.
authors.forename: Jae-Young
authors.initials: JY
authors.lastname: Jung
authors.affiliation: Research Animal Resource Center, Korea Institute of Science and Technology (KIST), Seoul 02792, Korea.
authors.forename: Seung Eun
authors.initials: SE
authors.lastname: Lee
authors.affiliation: Center for Neuroscience and Functional Connectomics, Brain Science Institute, Korea Institute of Science and Technology (KIST), Seoul 02792, Korea.; Neuroscience Program, University of Science and Technology (UST), Daejeon 34113, Korea.
authors.forename: Eun Mi
authors.initials: EM
authors.lastname: Hwang
authors.affiliation: Center for Neuroscience and Functional Connectomics, Brain Science Institute, Korea Institute of Science and Technology (KIST), Seoul 02792, Korea.; Neuroscience Program, University of Science and Technology (UST), Daejeon 34113, Korea.; KU-KIST School of Converging Science and Technology, Korea University, Seoul 02841, Korea.
authors.forename: C Justin
authors.initials: CJ
authors.lastname: Lee
keywords: DLX-cre; bestrophin-1; pSico; parvalbumin; thalamic reticular nucleus; virus