• the Korean Society for Brain and Neural Sciences


Original Article

Exp Neurobiol 2016; 25(3): 120-129

Published online June 30, 2016

© The Korean Society for Brain and Neural Sciences

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

Jae-Young Jung1,3, Seung Eun Lee2, Eun Mi Hwang1,3 and C. Justin Lee1,3,4*

1Center for Neuroscience and Functional Connectomics, Brain Science Institute, Korea Institute of Science and Technology (KIST), Seoul 02792, 2Research Animal Resource Center, Korea Institute of Science and Technology (KIST), Seoul 02792, 3Neuroscience Program, University of Science and Technology (UST), Daejeon 34113,4KU-KIST School of Converging Science and Technology, Korea University, Seoul 02841, Korea

Correspondence to: *To whom correspondence should be addressed.
TEL: 82-2-958-6940, FAX: 82-2-958-6919

Received: May 19, 2016; Revised: May 30, 2016; Accepted: May 31, 2016


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.

Keywords: bestrophin-1, thalamic reticular nucleus, pSico, virus, DLX-cre, parvalbumin