Laperrousaz, B., Porte, S., Gerbaud, S. et al. (6 more authors) (2018) Direct transfection of clonal organoids in matrigel microbeads : a promising approach toward organoid-based genetic screens. Nucleic Acids Research, 46 (12). e70. ISSN 0305-1048
Abstract
Organoid cultures in 3D matrices are relevant models to mimic the complex in vivo environment that supports cell physiological and pathological behaviors. For instance, 3D epithelial organoids recapitulate numerous features of glandular tissues including the development of fully differentiated acini that maintain apico-basal polarity with hollow lumen. Effective genetic engineering in organoids would bring new insights in organogenesis and carcinogenesis. However, direct 3D transfection on already formed organoids remains challenging. One limitation is that organoids are embedded in extracellular matrix and grow into compact structures that hinder transfection using traditional techniques. To address this issue, we developed an innovative approach for transgene expression in 3D organoids by combining single-cell encapsulation in Matrigel microbeads using a microfluidic device and electroporation. We demonstrate that direct electroporation of encapsulated organoids reaches up to 80% of transfection efficiency. Using this technique and a morphological read-out that recapitulate the different stages of tumor development, we further validate the role of p63 and PTEN as key genes in acinar development in breast and prostate tissues. We believe that the combination of controlled organoid generation and efficient 3D transfection developed here opens new perspectives for flow-based high-throughput genetic screening and functional genomic applications.
Metadata
Item Type: | Article |
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Authors/Creators: |
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Copyright, Publisher and Additional Information: | © 2018 The Authors. Published by Oxford University Press on behalf of Nucleic Acids Research. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com |
Keywords: | Cell biology; DNA-Mediated Cell Transformation and Nucleic Acids Transfer |
Dates: |
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Institution: | The University of Sheffield |
Academic Units: | The University of Sheffield > Faculty of Medicine, Dentistry and Health (Sheffield) > Department of Oncology (Sheffield) |
Depositing User: | Symplectic Sheffield |
Date Deposited: | 13 Nov 2019 11:46 |
Last Modified: | 16 Nov 2019 09:19 |
Status: | Published |
Publisher: | Oxford University Press (OUP) |
Refereed: | Yes |
Identification Number: | 10.1093/nar/gky030 |
Open Archives Initiative ID (OAI ID): | oai:eprints.whiterose.ac.uk:152206 |