Generation of BAC transgenic epithelial organoids

PLoS One. 2013 Oct 18;8(10):e76871. doi: 10.1371/journal.pone.0076871. eCollection 2013.

Abstract

Under previously developed culture conditions, mouse and human intestinal epithelia can be cultured and expanded over long periods. These so-called organoids recapitulate the three-dimensional architecture of the gut epithelium, and consist of all major intestinal cell types. One key advantage of these ex vivo cultures is their accessibility to live imaging. So far the establishment of transgenic fluorescent reporter organoids has required the generation of transgenic mice, a laborious and time-consuming process, which cannot be extended to human cultures. Here we present a transfection protocol that enables the generation of recombinant mouse and human reporter organoids using BAC (bacterial artificial chromosome) technology.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Chromosomes, Artificial, Bacterial / genetics*
  • Epithelium / growth & development
  • Epithelium / metabolism*
  • Genetic Engineering / methods
  • Green Fluorescent Proteins / genetics
  • Green Fluorescent Proteins / metabolism
  • Histones / genetics
  • Humans
  • Intestinal Mucosa / metabolism
  • Intestines / cytology
  • Luminescent Proteins / genetics
  • Luminescent Proteins / metabolism
  • Mice
  • Mice, Transgenic
  • Microscopy, Confocal
  • Organoids / cytology
  • Organoids / metabolism*
  • Tissue Culture Techniques / methods
  • Transfection / methods*
  • Transgenes / genetics
  • Tubulin / genetics
  • Video Recording / methods

Substances

  • Histones
  • Luminescent Proteins
  • Tubulin
  • red fluorescent protein
  • Green Fluorescent Proteins