Chapter title |
Lentivirus-Based Stable Gene Delivery into Intestinal Organoids.
|
---|---|
Chapter number | 2 |
Book title |
Gastrointestinal Physiology and Diseases
|
Published in |
Methods in molecular biology, January 2016
|
DOI | 10.1007/978-1-4939-3603-8_2 |
Pubmed ID | |
Book ISBNs |
978-1-4939-3601-4, 978-1-4939-3603-8
|
Authors |
Yoshiaki Maru M.D., Ph.D., Kaoru Orihashi, Yoshitaka Hippo, Yoshiaki Maru |
Editors |
Andrei I. Ivanov |
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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Other | 7 | 7% |
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