piggyBac transposon-mediated cellular transgenesis in mammalian forebrain by in utero electroporation.
piggyBac transposon-mediated cellular transgenesis in mammalian forebrain by in utero electroporation.
复制标题
DOI：
10.1101/pdb.prot073650
复制
发表时间：
2014-07-01
影响因子：
--
通讯作者：
LoTurco, Joseph J
中科院分区：
文献类型：
--
作者：
Chen, Fuyi;Maher, Brady J;LoTurco, Joseph J
子宫内电穿孔（IUE）是一种在体内将质粒DNA传递到哺乳动物神经祖细胞和新皮层神经元的有效转染方法。虽然IUE可以有效地将多个DNA质粒传递到细胞群体中，但不幸的是，传递到神经祖细胞中的质粒大部分仍然是次要的，并且经常在细胞分裂后失活或丢失。这导致了一种“出生日期”标记的形式，其中只有不经历第二次细胞分裂的细胞类型继续表达转染的质粒。这限制了IUE与标准质粒的应用，并排除了其在操作或标记祖细胞完整细胞系的实验中的使用。为了避免IUE中质粒的偶发性丢失，我们使用了一个二元piggyBac转座子系统来诱导转基因的非病毒基因组整合。这些转基因在细胞分裂后似乎不会失活，这导致神经元和胶质细胞稳定的体细胞转基因。与标准IUE一样，该系统可以与多种质粒组合一起使用，以实现多色标记和功能丧失和功能获得操作。在本协议中，我们描述了通过IUE传递二元piggyBac转座子质粒系统的方法。
In utero electroporation (IUE) is an effective transfection method for delivering plasmid DNA into neural progenitor cells and neurons of mammalian neocortex in vivo. Although IUE is effective at delivering multiple DNA plasmids into populations of cells, unfortunately plasmids delivered into neural progenitor cells remain largely episomal and often get inactivated or lost after cell division. This results in a form of "birthdate" labeling in which only the cell types that do not undergo a second cell division continue to express the transfected plasmids. This limits the application of IUE with standard plasmids and precludes its use in experiments where manipulating or labeling the complete cell lineage of a progenitor is desired. To circumvent this episomal loss of plasmid in IUE, we have used a binary piggyBac transposon system to induce nonviral genomic integration of transgenes. These transgenes do not appear to inactivate after cell division, and this results in stable somatic cellular transgenesis of neurons and glia. Like standard IUE, the system can be used with multiple combinations of plasmids to achieve multicolor labeling and both loss-of-function and gain-of-function manipulations. In this protocol, we describe the method for delivering a binary piggyBac transposon plasmid system by IUE.