Figure 1.

Genetic design of the porcine intestinal cancer model. (A) The inducible polycistronic oncogene cassette is a Sleeping Beauty transposon controlled by a CAG promoter. The cassette consists of two parts: The first part encodes a red fluorescent protein (RFP) reporter and a puromycin N‐acetyl transferase. Its transcription is terminated by a strong polyadenylation site, which prevents read‐through into the second part. Flp recombinase recognition target sites (FRT) flank the first part and enable excision. The second part consists of yellow fluorescent protein (YFP) and three oncogenes KRAS‐G12D, cMYC and SV40LT flanked by Dre recombinase recognition target sites (ROX). Expression from the oncogene cassette can be terminated by Dre recombination or inhibited using the doxycycline‐responsive element rtTR‐KRAB docking at the Tet‐responsive elements (TRE); (B) the FlpO‐inducible activator cassette is also a Sleeping Beauty transposon. Expression from the cassette is controlled by an intestinal‐specific villin promoter. The cassette encodes a blue fluorescent protein (BFP) reporter and a 4‐OHT‐inducible estrogen receptor‐type2‐conjugated Flp recombinase (Flp‐ERT2). Probe positions for Southern blotting are indicated in both cassette maps; (C) overview of the cloning procedure. Sleeping Beauty mediates stable integration of the cassettes, followed by porcine SCNT by handmade cloning and transfer of transgenic porcine blastocyst into surrogate landrace sows. Image of TG pigs at day 357; (D) Southern blot for quantification of cassettes in neonate TG minipig ear fibroblasts probed by either ³²P‐dCTP RFP DNA probe or (E) ³²P‐dCTP Flp DNA probe. Lanes 1–4 depict a 1‐kb ladder, WT fibroblast DNA as negative control, and WT fibroblast DNA spiked by either Flp activator‐ or oncogene cassette DNA as positive controls. Lanes 5–17 present genomic fibroblast DNA from 13 TG minipigs. High molecular bands in pigs no. 555 and 563 are likely artifacts. (F) Mapping of the genomic integration site of the oncogene cassette to a region between the genes NLGN1 and NAALADL2 on chromosome 13 using LDI‐PCR in TG minipig no. 556 fibroblasts.