Figure 4.

4‐OHT activation of the oncogene cassette in TG intestinal organoids (in vitro) and intestinal TG biopsies (ex vivo). (A) TG porcine intestinal organoids at days 0, 2, and 4 (with two distinct growth patterns); (B) quantification of oncogene cassettes and recombination events by ddPCR in 4‐OHT‐treated and untreated organoid cultures and intestinal biopsies. Shown are results of five to six biological replicates plotted as mean ± SE normalized to pig genome equivalents; (C) recombination validated by transition PCR in WT and TG organoid cultures (lanes 5–7) and intestinal biopsies (lanes 8–11). The transition PCR assay revealed recombination in all TG organoids and biopsies exposed to 4‐OHT, but in none of the untreated biopsies. NTC, WT fibroblasts, and TG fibroblasts +/− CAG‐Flp were included as controls (lanes 1–4); (D) Flp, RFP, and SV40LT transcript levels in TG organoids +/− 4‐OHT quantified by RT‐PCR.