A method for stable transgenesis of radial glia lineage in rat neocortex by piggyBac mediated transposition
- 作者：Fuyi Chen fuyi.chen@uconn.edu ; Joseph LoTurco ; joseph.loturco@uconn.edu
- 关键词：IUE ; in utero electroporation ; PB ; piggyBac transponson ; CNS ; central nervous system ; GFP ; green fluorescent protein ; RFP ; red fluorescent protein ; SVZ ; subventricular zone ; TR ; terminal repeats ; EGFR ; epidermal growth factor receptor ; iPS ; induced plurit
- 刊名：Journal of Neuroscience Methods
- 出版年：2012
- 期刊代码：33_01650270
- 类别：neu
- 出版时间：15 June, 2012
- 卷：207
- 期：2
- 页码：172-180
- 文件大小：2409 K
摘要
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.