Efficient genetic engineering of human intestinal organoids using electroporation

Nature Protocols
Masayuki FujiiToshiro Sato

Abstract

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.

Citations

May 6, 2016·Cellular and Molecular Life Sciences : CMLS·Joana F S PereiraPeter Jordan
Aug 16, 2016·Developmental Biology·Sebastian R MerkerDaniel E Stange
Oct 8, 2016·Frontiers in Pharmacology·Fangkun LiuWei Zhao
Oct 26, 2016·Nature Reviews. Gastroenterology & Hepatology·Julie G InMark Donowitz
Dec 16, 2016·PloS One·Irina A OkkelmanDmitri B Papkovsky
Mar 30, 2017·Nature·Mariko ShimokawaToshiro Sato
Dec 6, 2017·Advanced Healthcare Materials·Pradip Shahi ThakuriHossein Tavana
Apr 12, 2018·Tissue Engineering. Part C, Methods·Mitchell R LaddDavid J Hackam
Jan 18, 2018·The Journal of Experimental Medicine·Takashi KanayaHiroshi Ohno
May 16, 2018·Proceedings of the National Academy of Sciences of the United States of America·Changlong LiuPhillip J Buckhaults
Jul 7, 2018·International Journal of Cancer. Journal International Du Cancer·Ye-Lim ParkTae-You Kim
Jan 28, 2017·American Journal of Physiology. Gastrointestinal and Liver Physiology·Else Driehuis, Hans Clevers
Aug 2, 2019·The Journal of Biological Chemistry·Jinsong WeiBing Zhao
Dec 6, 2019·Tissue Engineering. Part C, Methods·Carolyn GosztylaDavid J Hackam
Feb 14, 2018·Science Translational Medicine·Brian J LeibowitzJian Yu
Mar 11, 2020·EMBO Molecular Medicine·Zdeněk DvořákSridhar Mani
Mar 15, 2020·Journal of Biomedical Materials Research. Part a·Jinjian HuangJianan Ren
Jul 3, 2020·Molecular Nutrition & Food Research·Àngela Casanova-MartíAnna Ardévol
Sep 1, 2020·Pest Management Science·Luc SweversJohn Vontas
Sep 18, 2020·Current Protocols in Immunology·Cayetano Pleguezuelos-ManzanoHans Clevers
Sep 24, 2020·The EMBO Journal·Maureen SpitMadelon M Maurice
Sep 30, 2020·Proceedings of the National Academy of Sciences of the United States of America·Marjoke F DebetsBenjamin Schumann
Jul 19, 2017·Annual Review of Virology·Mayumi K HollyJason G Smith
Oct 21, 2018·Scientific Reports·Paulius RuzgysSaulius Šatkauskas
Aug 28, 2020·Frontiers in Bioengineering and Biotechnology·Steven N SteinwayDeok-Ho Kim
Dec 28, 2017·Human Reproduction Update·João Pedro Alves-Lopes, Jan-Bernd Stukenborg
Dec 20, 2019·Nature Protocols·Chiara M CattaneoEmile E Voest
Apr 21, 2020·Wiley Interdisciplinary Reviews. Systems Biology and Medicine·Edwin K SilvermanJan Baumbach
Jan 18, 2019·Tissue Engineering. Part a·Mitchell R LaddDavid J Hackam

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