2013
DOI: 10.1002/9780470151808.sc05a06s27
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Retroviral Gene Expression Control in Primary Organoid Cultures

Abstract: In this unit, we describe a protocol for regulated gene expression in primary endodermal organoid culture using retroviral vectors. The study of gene function in endodermal epithelia such as those lining the stomach, small intestine, and colon has so far mainly relied on the generation of transgenic mouse lines. Establishing such animal models is laborious, expensive, and time‐consuming. Ever‐expanding endodermal organoids, grown in an in vitro 3‐D epithelial culture system, faithfully recapitulate the in vivo… Show more

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Cited by 31 publications
(25 citation statements)
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“…These studies demonstrate that human enteroids constitute a reliable human disease model with a possibility to move towards a personalized screening. Human enteroids can be genetically modified using DNA transfection or infection with viral particles 38 . This provides a powerful tool to study gene-specific functions within the human epithelial organoids or correct genetic mutations.…”
Section: Discussionmentioning
confidence: 99%
“…These studies demonstrate that human enteroids constitute a reliable human disease model with a possibility to move towards a personalized screening. Human enteroids can be genetically modified using DNA transfection or infection with viral particles 38 . This provides a powerful tool to study gene-specific functions within the human epithelial organoids or correct genetic mutations.…”
Section: Discussionmentioning
confidence: 99%
“…CRITICAL: Alternatives to mouse or human organoid infection with viral vectors include transfection 38 or electroporation 39 of plasmid DNA vectors.…”
Section: Methodsmentioning
confidence: 99%
“…In this article, we provide step-by-step protocols for mouse intestinal organoid generation (modified from Refs 36,37 ) lentiviral infection (modified from a retroviral transfection protocol in Ref 38 ), human CRC organoid generation (modified from Refs 15,39 ), and colonoscopy-guided mucosal injection (modified from Ref 32 and previously described in Refs 34,40 ) for CRC modeling. 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%
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“…Changes in gene expression can be readily effected in cultured organoids via transfection of DNA and small interfering RNA, or infection with recombinant retro- and lentiviruses 72 73. The successful development of organoid cryopreservation procedures also enables long-term storage of specific culture samples or batches.…”
Section: Intestinal Organoids As Experimental Toolsmentioning
confidence: 99%