A method for stable transgenesis of radial glia lineage in rat neocortex by piggyBac mediated transposition.
A method for stable transgenesis of radial glia lineage in rat neocortex by piggyBac mediated transposition.
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DOI：
10.1016/j.jneumeth.2012.03.016
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发表时间：
2012-06-15
影响因子：
3
通讯作者：
LoTurco J
中科院分区：
文献类型：
--
作者：
Chen F;LoTurco J
为了确定指定神经细胞类型的机制，需要将谱系追踪与细胞转基因相结合的方法。目前可用的方法包括病毒感染和cre介导重组。子宫内电穿孔（IUE）已被用于多种物种的神经祖细胞同时传递多个转基因。在标准的IUE中，大多数质粒仍然是外体的，在细胞分裂过程中丢失，因此转基因不会在完整的神经谱系中表达。在这里，我们将IUE与二元piggyBac转座子系统（PB-IUE）结合起来，并表明与传统的IUE不同，单胚胎转染带有piggyBac转座子系统的新皮质径向胶质细胞可在径向胶质细胞的神经谱系中稳定地表达转基因：皮质神经元、星形胶质细胞、少突胶质细胞和嗅球中间神经元。我们还开发了一个具有不同启动子的供体和辅助质粒的模块化工具包，允许在祖细胞亚群中进行shRNA、双链表达和突变。为了证明该工具的实用性，我们表明表皮生长因子受体（EGFR）的转基因扩大了祖细胞产生的星形胶质细胞和少突胶质细胞的数量。相对容易实现和实验的灵活性应该使piggyBac IUE方法成为跟踪和操纵神经谱系的有价值的新工具。
Methods that combine lineage tracing with cellular transgenesis are needed in order to determine mechanisms that specify neural cell types. Currently available methods include viral infection and Cre-mediated recombination. In utero electroporation (IUE) has been used in multiple species to deliver multiple transgenes simultaneously into neural progenitors. In standard IUE, most plasmids remain episomal, are lost during cell division, and so transgenes are not expressed in the complete neural lineage. Here we combine IUE with a binary piggyBac transposon system (PB-IUE), and show that unlike conventional IUE, a single embryonic transfection of neocortical radial glia with a piggyBac transposon system results in stable transgene expression in the neural lineage of radial glia: cortical neurons, astrocytes, oligodendrocytes, and olfactory bulb interneurons. We also developed a modular toolkit of donor and helper plasmids with different promoters that allows for shRNA, bicistronic expression, and trangenesis in subsets of progenitors. As a demonstration of the utility of the toolkit we show that transgenesis of epidermal growth factor receptor (EGFR) expands the number of astrocytes and oligodendrocyrtes generated from progenitors. The relative ease of implementation and experimental flexibility should make the piggyBac IUE method a valuable new tool for tracking and manipulating neural lineages.