Fig 1.
The Cre-inducible vector encoding KRASG12D and TP53R167H.
(a) Schematic diagram of the vector encoding Cre-inducible KRASG12D and TP53R167H. (b) PCR analysis for the presence of KRASG12D and TP53R167H in genomic DNA isolated from the indicated cloned offspring.
Fig 2.
Inducible expression of KRASG12D and TP53R167H is transforming and tumorigenic.
(a) RT-PCR analysis of KRASG12D and TP53R167H mRNA expression in the fibroblast cell lines from each of the 4 transgenic clones treated with AdCre or AdGFP. (b) Comparison of cell morphology between AdCre and untreated control cells in culture, stained with H&E. (c) Normalized MFU measured by FACS at time points following Carboxyfluorescein succinimidyl ester (CFSE) dye loading of cells. (d) Graphical analysis of the mean number of migrating cells from triplicate plating of each of the 4 cell lines. (e) Graphical analysis of the mean number of colonies growing in soft agar for each cell line from triplicate plating. (c-e: all data points are the mean of the 4 cell lines derived from pigs 63–1, 63–2, 63–3, 63–4; error bars = SD; *p-value ≤ 0.05; **p-value ≤ 0.01).
Table 1.
Induction of Tumors Following Injection of AdCre or AdGFP treated Oncopig Transgenic Cells into Immunocompromised Mice (Days to 2000 mm3).
Fig 3.
Tumors arose from each AdCre treated cell line injected into immune compromised mice.
Similar tumors (a) developed from all AdCre cell lines and no tumors developed from AdGFP cell injections; Histological analysis (b) revealed the tumors to be densely cellular non encapsulated and infiltrative neoplasm. 10x and insert 40X H&E Stain.
Fig 4.
Inducible expression of KRASG12D and TP53R167H is tumorigenic in transgenic pigs.
(a–c) Ultrasound images of tumors developing 10 days after, and (d–f) images of these tumors at necropsy 20 days after intramuscular (IM, Pig 1), subcutaneous (SQ, Pig 3), or intra-testicular (IT, Pig 1) injection of AdCre in transgenic oncopigs containing a Cre-inducible vector encoding KRASG12D and TP53R167H; (g–i) H&E stained sections show the tumor and adjacent normal tissue (2x) with an inserted high magnification photo (20x).
Fig 5.
Injection of AdGFP into transgenic pigs did not induce tumors.
The lack of any detectable changes to the injection site on d20 post- injection is shown at the surface (a–c); in underlying tissue (d–f); or upon histological examination (g–i).
Table 2.
Summary of porcine tumor growth in vivo.