Efficient genetic engineering of human intestinal organoids using electroporation
Efficient genetic engineering of human intestinal organoids using electroporation
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DOI：
10.1038/nprot.2015.088
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发表时间：
2015-10-01
期刊：
影响因子：
14.8
通讯作者：
Sato, Toshiro
中科院分区：
文献类型：
--
作者：
Fujii, Masayuki;Matano, Mami;Sato, Toshiro
在未转化的人肠道细胞中进行基因修饰是研究肠道疾病基因功能的一种有吸引力的方法。然而，由于缺乏实用的工具，这些研究在很大程度上依赖于替代物，如基因工程小鼠或永生化的人类细胞系。通过利用最近开发的肠道类器官培养方法，我们开发了一种通过基因载体的电穿孔来调节未转化的人结肠类器官中感兴趣的基因的方法。在这里，我们描述了一个详细的协议，通过培养与基本生长因子在基底膜基质肠类器官的产生。我们还描述了如何通过piggyBac转座子稳定整合基因，以及使用CRCRISPRSPR-Cas9系统进行精确的基因组编辑。从人类结肠样品的隐窝分离开始，可以在3周内获得遗传修饰的类器官。
Gene modification in untransformed human intestinal cells is an attractive approach for studying gene function in intestinal diseases. However, because of the lack of practical tools, such studies have largely depended upon surrogates, such as gene-engineered mice or immortalized human cell lines. By taking advantage of the recently developed intestinal organoid culture method, we developed a methodology for modulating genes of interest in untransformed human colonic organoids via electroporation of gene vectors. Here we describe a detailed protocol for the generation of intestinal organoids by culture with essential growth factors in a basement membrane matrix. We also describe how to stably integrate genes via the piggyBac transposon, as well as precise genome editing using the CRCRISPRSPRSPR-Cas9 system. Beginning with crypt isolation from a human colon sample, genetically modified organoids can be obtained in 3 weeks.