Efficient and simple genetic engineering of enteroids using mouse isolated crypts for investigating intestinal functions

Intestinal epithelial cells separate subepithelial tissues from luminal environment formed with food, incoming pathogens, and resident intestinal microbiota, etc., and elicit various intestinal function. Enteroid, a three-dimensional culture system of small intestinal epithelial cells, has been wide...

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Bibliographic Details
Published in:Biochemical and biophysical research communications 2022-12, Vol.637, p.153-160
Main Authors: Ohira, Shuya, Yokoi, Yuki, Ayabe, Tokiyoshi, Nakamura, Kiminori
Format: Article
Language:English
Subjects:
Animals
Animals, Genetically Modified
Cell Count
Defecation
Gene transfer
Genetic Engineering
Genetic Therapy
Isolated crypt
Mice
Small intestinal epithelial cell
Stem cell niche
Transgenic enteroid
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Summary:Intestinal epithelial cells separate subepithelial tissues from luminal environment formed with food, incoming pathogens, and resident intestinal microbiota, etc., and elicit various intestinal function. Enteroid, a three-dimensional culture system of small intestinal epithelial cells, has been widely used for analyzing the intestinal function, further a transgenic enteroid was developed to investigate the molecular mechanisms. However, conventional transgenic enteroid production method, which transfer gene into single stem cells, has limitations including low efficiency and time-consuming. Here we show that by gene transfer into small intestinal isolated crypts maintaining stem cell niche, a transgenic enteroid was obtained quickly and efficiently. Isolated crypts were transfected by lentiviral vector without separating into single cells, and transgenic enteroid composed of all lineages of intestinal epithelial cells was generated at day 7 with yield of 56%, maintaining the intestinal function in drug transport and innate immunity. Our efficient and simple transgenic enteroid generation method enables high-throughput investigation of intestinal epithelial cells and contributes to understanding intestinal function. •Efficient and simple method for generating transgenic enteroid was established.•Gene was transferred in intestinal epithelial stem cell (IESC)s of isolated crypts.•Transgenic enteroids maintain transgene expression in passage culture over a month.•Transgenic IESCs retain multipotency to differentiate into all lineages.•Terminally differentiated cells from transgenic IESCs maintain intact function.
ISSN:0006-291X
1090-2104
DOI:10.1016/j.bbrc.2022.11.008