Efficient genetic engineering of human intestinal organoids using electroporation

Fujii, Masayuki; Matano, Mami; Nanki, Kosaku; Sato, Toshiro

doi:10.1038/nprot.2015.088

Cited by 271 publications

(258 citation statements)

References 12 publications

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“…Using the growth factors first described by Sato et al 11,40 , enteroids and/or colonoids can be produced from each segment of the small intestine (duodenum, jejunum and ileum), colon (proximal, transverse, distal), and recto-sigmoid. These cultures grown ex vivo have been maintained in various laboratories for >2 years with no detectable changes in chromosomal number or other cancerous characteristics of wild-type cultures 41 .…”

Section: Human Ex Vivo Modelsmentioning

confidence: 99%

Human mini-guts: new insights into intestinal physiology and host–pathogen interactions

In¹,

Foulke‐Abel²,

Estes³

et al. 2016

Nat Rev Gastroenterol Hepatol

103

117

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The development of indefinitely propagating human ‘mini-guts’ has led to a rapid advance in gastrointestinal research related to transport physiology, developmental biology, pharmacology, and pathophysiology. These mini-guts, also called enteroids or colonoids, are derived from LGR5+ intestinal stem cells isolated from the small intestine or colon. Addition of WNT3A and other growth factors promotes stemness and results in viable, physiologically functional human intestinal or colonic cultures that develop a crypt–villus axis and can be differentiated into all intestinal epithelial cell types. The success of research using human enteroids has highlighted the limitations of using animals or in vitro, cancer-derived cell lines to model transport physiology and pathophysiology. For example, curative or preventive therapies for acute enteric infections have been limited, mostly due to the lack of a physiological human intestinal model. However, the human enteroid model enables specific functional studies of secretion and absorption in each intestinal segment as well as observations of the earliest molecular events that occur during enteric infections. This Review describes studies characterizing these human mini-guts as a physiological model to investigate intestinal transport and host pathogen interactions.

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Section: Human Ex Vivo Modelsmentioning

confidence: 99%

Human mini-guts: new insights into intestinal physiology and host–pathogen interactions

In¹,

Foulke‐Abel²,

Estes³

et al. 2016

Nat Rev Gastroenterol Hepatol

103

117

View full text Add to dashboard Cite

show abstract

“…In details, the locus of the cystic fibrosis transmembrane conductor receptor in cultured intestinal stem cells derived from cystic fibrosis patients was accurately repaired using CRISPR/Cas9 genome editing system via homologous recombination [97]. Efficient gene transfer was also successfully established in human intestinal organoids by this platform [96]. Human pluripotent stem cells (hPSCs) such as induced pluripotent stem cells (iPSCs) and embryonic stem cells (ESCs) are considered as very useful tools for elucidating regulatory processes during early development and the pathogenesis of genetic disorders [100, 101].…”

Section: Conclusion Remarks and Future Perspectivesmentioning

confidence: 99%

Brain tumor modeling using the CRISPR/Cas9 system: state of the art and view to the future

et al. 2016

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Although brain tumors have been known tremendously over the past decade, there are still many problems to be solved. The etiology of brain tumors is not well understood and the treatment remains modest. There is in great need to develop a suitable brain tumor models that faithfully mirror the etiology of human brain neoplasm and subsequently get more efficient therapeutic approaches for these disorders. In this review, we described the current status of animal models of brain tumors and analyzed their advantages and disadvantages. Additionally, prokaryotic clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9), a versatile genome editing technology for investigating the functions of target genes, and its application were also introduced in our present work. We firstly proposed that brain tumor modeling could be well established via CRISPR/Cas9 techniques. And CRISPR/Cas9-mediated brain tumor modeling was likely to be more suitable for figuring out the pathogenesis of brain tumors, as CRISPR/Cas9 platform was a simple and more efficient biological toolbox for implementing mutagenesis of oncogenes or tumor suppressors that were closely linked with brain tumors.

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“…Mouse or human intestinal organoid infection with viral vectors can be substituted with transfection 38 or electroporation of plasmid DNA vectors 41 .…”

Section: Introductionmentioning

confidence: 99%

Colonoscopy-based colorectal cancer modeling in mice with CRISPR–Cas9 genome editing and organoid transplantation

et al. 2018

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Most genetically engineered mouse models of colorectal cancer are limited by tumor formation in the small intestine, a high tumor burden that limits metastasis, and the need to generate and cross mutant mice. Cell line or organoid transplantation models generally produce tumors in ectopic locations, such as the subcutaneous space, kidney capsule, or cecal wall, that do not reflect the native stromal environment of the colon mucosa. Here, we describe detailed protocols to rapidly and efficiently induce site-directed tumors in the distal colon of mice that are based on colonoscopy-guided mucosal injection. These techniques can be adapted to deliver viral vectors carrying Cre recombinase, CRISPR-Cas9 components, CRISPR-engineered mouse tumor organoids, or human cancer organoids to mice to model the adenoma-carcinoma-metastasis sequence of tumor progression. The colonoscopy injection procedure takes approximately 15 minutes, including preparation. In our experience, anyone with reasonable hand-eye coordination can become proficient with basic mouse colonoscopy with a few hours of practice. These approaches are ideal for a wide range of applications, including assessment of gene function in tumorigenesis, examination of tumor-stroma interactions, studies of cancer metastasis, and translational research with patient-derived cancers.

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Efficient genetic engineering of human intestinal organoids using electroporation

Cited by 271 publications

References 12 publications

Human mini-guts: new insights into intestinal physiology and host–pathogen interactions

Human mini-guts: new insights into intestinal physiology and host–pathogen interactions

Brain tumor modeling using the CRISPR/Cas9 system: state of the art and view to the future

Colonoscopy-based colorectal cancer modeling in mice with CRISPR–Cas9 genome editing and organoid transplantation

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