Porlan Eva, Martí-Prado Beatriz, Consiglio Antonella, Fariñas Isabel
Cell Division and Cancer Group, Spanish National Cancer Research Centre (CNIO).
Centro de Investigaciones Biomédicas en Red sobre Enfermedades Neurodegenerativas (CIBERNED); Departmento de Biologìa Celular, Universidad de Valencia.
J Vis Exp. 2016 Feb 17(108):53282. doi: 10.3791/53282.
Relatively quiescent somatic stem cells support life-long cell renewal in most adult tissues. Neural stem cells in the adult mammalian brain are restricted to two specific neurogenic niches: the subgranular zone of the dentate gyrus in the hippocampus and the ventricular-subventricular zone (V-SVZ; also called subependymal zone or SEZ) in the walls of the lateral ventricles. The development of in vivo gene transfer strategies for adult stem cell populations (i.e. those of the mammalian brain) resulting in long-term expression of desired transgenes in the stem cells and their derived progeny is a crucial tool in current biomedical and biotechnological research. Here, a direct in vivo method is presented for the stable genetic modification of adult mouse V-SVZ cells that takes advantage of the cell cycle-independent infection by LVs and the highly specialized cytoarchitecture of the V-SVZ niche. Specifically, the current protocol involves the injection of empty LVs (control) or LVs encoding specific transgene expression cassettes into either the V-SVZ itself, for the in vivo targeting of all types of cells in the niche, or into the lateral ventricle lumen, for the targeting of ependymal cells only. Expression cassettes are then integrated into the genome of the transduced cells and fluorescent proteins, also encoded by the LVs, allow the detection of the transduced cells for the analysis of cell autonomous and non-autonomous, niche-dependent effects in the labeled cells and their progeny.
相对静止的体细胞干细胞支持大多数成体组织中的终生细胞更新。成年哺乳动物大脑中的神经干细胞局限于两个特定的神经发生微环境：海马齿状回的颗粒下区和侧脑室壁的室管膜下区（V-SVZ；也称为室管膜下区或SEZ）。开发针对成体干细胞群体（即哺乳动物大脑中的干细胞群体）的体内基因转移策略，使所需转基因在干细胞及其衍生后代中实现长期表达，是当前生物医学和生物技术研究中的一项关键工具。在此，我们提出一种直接的体内方法，用于对成年小鼠V-SVZ细胞进行稳定的基因改造，该方法利用了慢病毒（LVs）不依赖细胞周期的感染以及V-SVZ微环境高度特化的细胞结构。具体而言，当前方案包括将空的慢病毒（对照）或编码特定转基因表达盒的慢病毒注射到V-SVZ本身，以在体内靶向微环境中的所有类型细胞，或者注射到侧脑室腔中，仅靶向室管膜细胞。然后，表达盒被整合到转导细胞的基因组中，慢病毒编码的荧光蛋白可用于检测转导细胞，以便分析标记细胞及其后代中的细胞自主和非自主的、依赖微环境的效应。
