Abstract
Lentivirus-based gene delivery works efficiently for the majority of mammalian cells cultured under standard two-dimensional conditions. By contrast, intestinal epithelial organoids embedded into three-dimensional extracellular matrix appear to be resistant to lentiviral transduction. We observed that Matrigel, a matrix that reconstitutes a basement membrane and is indispensable for cell survival and proliferation, prevents lentiviruses from binding to intestinal cells. In this chapter, we describe a simple method of a highly efficient gene transduction into intestinal organoids. This method involves organoid dispersion into single intestinal epithelial cells, mixing these individual cells with lentiviral particles, plating on Matrigel, and subsequent re-embedding into Matrigel. Under these conditions, the majority of the cells are exposed to the virus in the absence of the matrix barrier while remaining attached to the matrix. Using a GFP-labeled lentivirus, we demonstrate that this method allows for highly efficient infection of intestinal organoids after overnight incubation of Matrigel-attached cells with lentiviral particles.
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Acknowledgment
This work was supported by Grants-in-Aid for Scientific Research (B) 26290044 from the Japan Society for the Promotion of Science (JSPS).
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Maru, Y., Orihashi, K., Hippo, Y. (2016). Lentivirus-Based Stable Gene Delivery into Intestinal Organoids. In: Ivanov, A. (eds) Gastrointestinal Physiology and Diseases. Methods in Molecular Biology, vol 1422. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-3603-8_2
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DOI: https://doi.org/10.1007/978-1-4939-3603-8_2
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Publisher Name: Humana Press, New York, NY
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Online ISBN: 978-1-4939-3603-8
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