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Efficient genetic engineering of human intestinal organoids using electroporation

Nature Protocols volume 10, pages 1474–1485 (2015)Cite this article

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An Author Correction to this article was published on 05 December 2018

This article has been updated

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.

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Figure 1: An overview of gene delivery to human intestinal organoids.
Figure 2: Organoid-forming competency of single-cell dissociated organoids.
Figure 3: Comparison of gene-delivery methods and conditions.
Figure 4: An experimental protocol for electroporation to human intestinal organoids.
Figure 5: Flow cytometry analysis of GFP expression after delivery of a GFP-expressing piggyBac vector with optimized electroporation protocol.
Figure 6: Images of the organoids during selection.

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Change history

  • 05 December 2018

    The version of this paper originally published shows incorrect units for two plasmid concentrations. In the "Reagent Setup" section, the instructions for sgRNA-Cas9 plasmid should read "Adjust the concentration of each plasmid to 1 μg μl–1,” rather than "to 1 μg ml–1.” Similarly, all concentrations in the tables in Steps 49A, 49C, and 49D should be in μg μl–1 instead of μg ml–1. Please note that these units have not been corrected in the PDF and HTML versions of the protocol available online.

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Acknowledgements

This work was supported by grants from a research program of the Project for Development of Innovative Research on Cancer Therapeutics (P-Direct), by a Grant-in-Aid for Scientific Research on Innovative Areas 'Stem Cell Aging and Disease' and by Grants-in-Aid for Scientific Research, Ministry of Education, Culture, Sports, Science and Technology of Japan. L-Wnt3A cells and the R-spondin1–producing cell line were kindly gifted by H. Clevers (Hubrecht Institute) and C. Kuo (Stanford University), respectively.

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Author notes

  1. Masayuki Fujii and Mami Matano: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Gastroenterology, Keio University School of Medicine, Tokyo, Japan

    Masayuki Fujii, Mami Matano, Kosaku Nanki & Toshiro Sato

  2. Department of Surgical Oncology, The University of Tokyo, Tokyo, Japan

    Masayuki Fujii

Authors
  1. Masayuki Fujii
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  2. Mami Matano
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  3. Kosaku Nanki
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  4. Toshiro Sato
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Contributions

M.F., M.M. and K.N. performed the experiments. T.S. conceived and designed the project. M.F. wrote the manuscript.

Corresponding author

Correspondence to Toshiro Sato.

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Competing interests

T.S. is an inventor on patents involving the organoid culture system (WO/2010/090513 and WO/2012/168930).

Integrated supplementary information

Supplementary Figure 1 Reduced growth efficiency by continuous treatment with CHIR99021.

Images of organoids treated with CHIR99021 for 7 days (left) and without CHIR99021 (with Wnt3A and R-Spondin1)(right) after single cell dissociation. Scale bars, 500 µm.

Supplementary Figure 2 Diagram of the comparisons for optimization of the electroporation method.

The conditions with red characters are superior to the others, and the protocol was optimized by combination of these conditions.

Supplementary Figure 3 Effect of filtration of the dissociated organoids.

Images of dissociated organoids before (left) and after (right) passing through a filter with 20-µm pores. Scale bars, 200 µm.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–3 (PDF 724 kb)

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Fujii, M., Matano, M., Nanki, K. et al. Efficient genetic engineering of human intestinal organoids using electroporation. Nat Protoc 10, 1474–1485 (2015). https://doi.org/10.1038/nprot.2015.088

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