Generation of BAC transgenic epithelial organoids

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.

Figure 1. BAC transgenic mouse intestinal organoids.

(A) Tagging cassettes for BAC recombineering. Upper panel illustrates the C-terminal GFP-tagging cassette with a neomycin resistance gene downstream of an IRES sequence, which was used in to tag histone H2A and TUBB5. Lower panel shows the C-terminal tagRFP tagging cassette with a neomycine resistance gene downstream of a PGK promoter and flanked by loxP sites, which was used to tag the lysozyme gene. gb3: bacterial promoter, PGK: phosphoglycerate kinase promoter, IRES: internal ribosome entry site. (B) FACS analysis of mouse intestinal organoids 48 h after transfection with the pmax-GFP plasmid. Middle panel shows cell viability using propidium iodide (PI), right panel shows the percentage of GFP transfected cells. (C) Section of a well with mouse organoids 48 h after H2A-GFP BAC transfection using lipofectamine. Arrow points to a successfully transfected cell. Fluorescence images of a (D) H2A-GFP BAC transgenic organoid, (E) TUBB5-GFP BAC transgenic organoid, and (F) Lysozyme-tagRFP BAC transgenic organoid.

Figure 2. BAC transgenic human intestinal organoids.

(A) FACS analysis of human intestinal organoids 48 h after transfection with the pmax-GFP plasmid. Middle panel shows cell viability using propidium iodide (PI), right panel shows the percentage of GFP transfected cells. (B) Section of a well with human organoids 48 h after H2A-GFP BAC transfection with lipofectamine. Arrows point to successfully transfected cells. (C) Fluorescence image of a H2A-GFP BAC transgenic human organoid.

Figure 3. Snapshots of BAC transgenic organoid movies.

(A) a 36 h movie (2.4 frames/hour) of a growing mouse H2A BAC transgenic organoid, (B) a 51 h movie (2.2 frames/hour) of a growing mouse TUBB5-GFP BAC transgenic organoid, and (C) a 77 h movie (1 frame/hour) of a growing mouse Lysozyme-tagRFP BAC transgenic organoid.