Jung Jae-Young, Lee Seung Eun, Hwang Eun Mi, Lee C Justin
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.
Research Animal Resource Center, Korea Institute of Science and Technology (KIST), Seoul 02792, Korea.
Exp Neurobiol. 2016 Jun;25(3):120-9. doi: 10.5607/en.2016.25.3.120. Epub 2016 Jun 22.
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.
通过使用细胞类型特异性基因操作可以实现对特定基因的细胞类型表达模式的评估。最近，依赖于cre重组酶的基因沉默工具pSico在神经科学研究中变得很流行。然而，pSico有一个关键限制，由于绿色荧光蛋白（GFP）报告基因的切除，基因沉默细胞无法通过荧光进行识别。为了克服这一限制，我们新开发了pSico-Red，在两个loxP位点之外以mCherry基因作为报告基因，这样在所有转染细胞中都能检测到红色mCherry信号。当细胞表达cre时，GFP被切除且shRNA被激活，导致GFP消失。pSico-Red的这一特性不仅提供了细胞类型特异性基因沉默，还能识别表达cre的细胞。利用该系统，我们首次证明了Bestrophin-1（Best1）在丘脑网状核（TRN）中的神经元表达以及TRN神经元特异性的Best1基因沉默。我们将携带Best1-shRNA的腺相关病毒（AAV）与在远端同源盒5/6（DLX5/6）启动子下表达cre的转基因小鼠相结合，DLX5/6是抑制性神经元的标志物。首先，我们通过免疫组织化学发现TRN中几乎所有的抑制性神经元都表达Best1。使用pSico-Red病毒，我们发现80%被感染的TRN神经元是DLX5/6-cre阳性但小白蛋白阴性。最后，我们发现Best1-shRNA显著降低了DLX5/6-cre阳性神经元中的Best1。我们的研究表明TRN神经元强烈表达Best1，并且pSico-Red是用于细胞类型特异性基因操作和识别特定细胞群体的有价值工具。
