期刊
NATURE PROTOCOLS
卷 10, 期 10, 页码 1474-1485出版社
NATURE PUBLISHING GROUP
DOI: 10.1038/nprot.2015.088
关键词
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资金
- Project for Development of Innovative Research on Cancer Therapeutics (P-Direct)
- Ministry of Education, Culture, Sports, Science and Technology of Japan
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.
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