Division of Brain Sciences, Keio University School of Medicine, Tokyo, Japan.
Institute of Cellular Neurosciences, Medical Faculty, University of Bonn, Bonn, Germany.
Glia. 2023 Feb;71(2):317-333. doi: 10.1002/glia.24273. Epub 2022 Sep 27.
Nerve/glial antigen 2 (NG2) is a protein marker of NG2 glia and mural cells, and NG2 promoter activity is utilized to target these cells. However, the NG2 promoter cannot target NG2 glia and mural cells separately. This has been an obstacle for NG2 glia-specific manipulation. Here, we developed transgenic mice in which either cell type can be targeted using the NG2 promoter. We selected a tetracycline-controllable gene induction system for cell type-specific transgene expression, and generated NG2-tetracycline transactivator (tTA) transgenic lines. We crossed tTA lines with the tetO-ChR2 (channelrhodopsin-2)-EYFP line to characterize tTA-dependent transgene induction. We isolated two unique NG2-tTA mouse lines: one that induced ChR2-EYFP only in mural cells, likely due to the chromosomal position effect of NG2-tTA insertion, and the other that induced it in both cell types. We then applied a Cre-mediated set-subtraction strategy to the latter case and eliminated ChR2-EYFP from mural cells, resulting in NG2 glia-specific transgene induction. We further demonstrated that tTA-dependent ChR2 expression could manipulate cell function. Optogenetic mural cell activation decreased cerebral blood flow, as previously reported, indicating that tTA-mediated ChR2 expression was sufficient to impact cellular function. ChR2-mediated depolarization was observed in NG2 glia in acute hippocampal slices. In addition, ChR2-mediated depolarization of NG2 glia inhibited their proliferation but promoted their differentiation in juvenile mice. Since the tTA-tetO combination is expandable, the mural cell-specific NG2-tTA line and the NG2 glia-specific NG2-tTA line will permit us to conduct observational and manipulation studies to examine in vivo function of these cells separately.
神经胶质抗原 2（NG2）是 NG2 胶质细胞和壁细胞的蛋白标志物，NG2 启动子活性被用于靶向这些细胞。然而，NG2 启动子不能分别靶向 NG2 胶质细胞和壁细胞。这一直是 NG2 胶质细胞特异性操作的障碍。在这里，我们开发了转基因小鼠，其中使用 NG2 启动子可以分别靶向这两种细胞类型。我们选择了一种四环素可控基因诱导系统用于细胞类型特异性转基因表达，并生成了 NG2-四环素转录激活子（tTA）转基因系。我们将 tTA 系与 tetO-ChR2（通道视紫红质-2）-EYFP 系杂交，以表征 tTA 依赖性转基因诱导。我们分离了两种独特的 NG2-tTA 小鼠系：一种仅在壁细胞中诱导 ChR2-EYFP，可能是由于 NG2-tTA 插入的染色体位置效应，另一种在两种细胞类型中诱导它。然后，我们在后一种情况下应用 Cre 介导的集合减法策略，并从壁细胞中消除 ChR2-EYFP，导致 NG2 胶质细胞特异性转基因诱导。我们进一步证明，tTA 依赖性 ChR2 表达可以操纵细胞功能。光遗传学壁细胞激活如先前报道的那样降低脑血流，表明 tTA 介导的 ChR2 表达足以影响细胞功能。在急性海马切片中观察到 ChR2 介导的 NG2 胶质细胞去极化。此外，ChR2 介导的 NG2 胶质细胞去极化抑制了它们在幼年小鼠中的增殖，但促进了它们的分化。由于 tTA-tetO 组合是可扩展的，壁细胞特异性 NG2-tTA 系和 NG2 胶质细胞特异性 NG2-tTA 系将允许我们进行观察和操作研究，以分别研究这些细胞的体内功能。
