Margaret M. Dyson Vision Research Institute, Department of Ophthalmology, Weill Medical College of Cornell University, New York, New York 10065, USA.
J Comp Neurol. 2010 Aug 15;518(16):3327-42. doi: 10.1002/cne.22402.
Previous studies showed that Tctex-1 immunoreactivity is selectively enriched in the germinal zones of adult brain. In this report we identify a regulatory region of the Tctex-1 gene that is capable of directing transgenic expression of green fluorescent protein (GFP) reporter that recapitulates the spatial and temporal expression pattern of endogenous Tctex-1. This construct specifically targeted expression to the nestin(+)/Pax6(+)/GLAST(+) radial glial cells and Tbr2(+) intermediate progenitors when the reporter construct was delivered to developing mouse neocortex via in utero electroporation. Characterization of mice transgenically expressing GFP under the same regulatory element showed that the GFP expression is faithful to endogenous Tctex-1 at the subgranular zone (SGZ) of dentate gyrus, ventricular/subventricular zone of lateral ventricles, and ependymal layer of 3rd ventricle of adult brains. Immunolocalization and bromodeoxyuridine incorporation studies of adult SGZ in four independent mouse lines showed that Tctex-1:GFP reporter selectively marks nestin(+)/GFAP(+)/Sox2(+) neural stem-like cells in two mouse lines (4 and 13). In two other mouse lines (17 and 18), Tctex-1:GFP is selectively expressed in Type-2 and Type-3 transient amplifying progenitors and a small subset of young neuronal progeny. The P/E-Tctex-1 reporter mouse studies independently confirmed the specific enrichment of Tctex-1 at adult SGZ stem/progenitor cells. Furthermore, these studies supported the notion that an analogous transcriptional program may be used to regulate neurogenesis in embryonic cerebral cortex and adult hippocampus. Finally, the genomic sequences and the reporter mouse lines described here provide useful experimental tools to advance adult neural stem cell research.
先前的研究表明,Tctex-1 免疫反应性在成人脑的生发区选择性富集。在本报告中,我们鉴定了 Tctex-1 基因的一个调节区,该区域能够指导绿色荧光蛋白(GFP)报告基因的转基因表达,该报告基因重现了内源性 Tctex-1 的时空表达模式。当报告基因构建体通过子宫内电穿孔递送至发育中的小鼠新皮层时,该构建体特异性地将表达靶向 nestin(+)/Pax6(+)/GLAST(+) 放射状胶质细胞和 Tbr2(+) 中间祖细胞。转 GFP 的小鼠的特征分析表明,GFP 表达在成年大脑的齿状回颗粒下区(SGZ)、侧脑室的室下区/室管膜区和第三脑室的室管膜层与内源性 Tctex-1 一致。对四个独立的小鼠系中成年 SGZ 的免疫定位和溴脱氧尿苷掺入研究表明,Tctex-1:GFP 报告基因选择性地标记了两个小鼠系中的 nestin(+)/GFAP(+)/Sox2(+) 神经样干细胞(4 和 13)。在另外两个小鼠系(17 和 18)中,Tctex-1:GFP 选择性地表达在 Type-2 和 Type-3 短暂扩增祖细胞和一小部分年轻的神经元祖细胞中。P/E-Tctex-1 报告基因小鼠研究独立证实了 Tctex-1 在成年 SGZ 干细胞/祖细胞中的特异性富集。此外,这些研究支持了这样一种观点,即类似的转录程序可能用于调节胚胎大脑皮层和成年海马体的神经发生。最后,这里描述的基因组序列和报告基因小鼠系为推进成年神经干细胞研究提供了有用的实验工具。