Department of Gastroenterology, Keio University School of Medicine, Tokyo, Japan.
Department of Surgical Oncology, The University of Tokyo, Tokyo, Japan.
Nat Protoc. 2015 Oct;10(10):1474-85. doi: 10.1038/nprot.2015.088. Epub 2015 Sep 3.
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 CRISPR-Cas9 system. Beginning with crypt isolation from a human colon sample, genetically modified organoids can be obtained in 3 weeks.
在未转化的人类肠道细胞中进行基因修饰是研究肠道疾病中基因功能的一种有吸引力的方法。然而，由于缺乏实用工具，此类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细胞系。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电穿孔基因载体在未转化的人结肠类器官中调节目的基因的方法。在这里，我们描述了一个详细的方案，即用基本生长因子在基底膜基质中培养来生成肠道类器官。我们还描述了如何通过 piggyBac 转座子稳定整合基因，以及使用 CRISPR-Cas9 系统进行精确的基因组编辑。从人结肠样本中分离隐窝开始，经过 3 周即可获得基因修饰的类器官。
