Efficient genetic engineering of human intestinal organoids using electroporation.
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.
Department of Gastroenterology, Keio University School of Medicine, Tokyo, Japan.|Department of Surgical Oncology, The University of Tokyo, Tokyo, Japan.
Masayuki
M
Fujii
Department of Gastroenterology, Keio University School of Medicine, Tokyo, Japan.
Mami
M
Matano
Department of Gastroenterology, Keio University School of Medicine, Tokyo, Japan.
Kosaku
K
Nanki
Department of Gastroenterology, Keio University School of Medicine, Tokyo, Japan.
Toshiro
T
Sato
D000818:Animals; D018929:Cell Culture Techniques; D002460:Cell Line; D018274:Electroporation; D005434:Flow Cytometry; D005818:Genetic Engineering / Q000379:methods*; D006801:Humans; D007422:Intestines / Q000166:cytology*; D051379:Mice; D009940:Organoids / Q000166:cytology*