Characterization of an inducible transgenic p53/Kras oncopig model for cancer

L.A. Rund, T. Collares, F. Seixas, K. Begnini, C.M. Counter, L.B. Schook
American Association for Cancer Research Annual Meeting, April 5-9, 2014, San Diego, CA


Due to the urgent need for more relevant animal models of cancer, a transgenic porcine model has been developed. Swine were chosen because of their genetic and physiological similarities with humans. The transgene construct contains oncogenic KRASG12D and dominant-negative p53R167H, downstream of a LoxP-polyA (STOP)-LoxP sequence (LSL) and a CAG promoter. These mutations were used because both KRASG12D, associated with increased cell motility and invasiveness and p53 R175H, a necessary tumor suppressor, occur at a high frequency in many types of human cancer. This design enables both temporal and spatial control of transgene expression. Cloned animals with this transgenic construct were produced by somatic cell nuclear transfer and 2 have reached 1.5 years of age with no development of tumors or any other abnormalities demonstrating that the transgene expression remains suppressed without cre-recombination and that no other difficulties have been produced by the cloning procedure. We have previously reported Cre recombination induced tumorgenicity of fibroblast lines derived from the clones. To date, the Ad-GFP-Cre treated cell lines (CRE) have been passaged more than 95 times at a dilution of 1:3 or above with no evidence of replicative senescence, while untreated and Ad-GFP treated transgenic cell lines (GFP) senesced by 45 passages. The clones have sired transgenic offspring from which fibroblast and keratinocyte cell lines have been derived. To verify that the tumorigenic phenotype was transmitted to offspring, both cell lineages were infected with Ad-GFP-Cre or Ad-GFP. Cre treatment induced morphological changes in both fibroblasts and keratinocytes, which included cell rounding, decreased cell size and diminished cell attachment to the culture dishes. All characteristics were first noted within 3 days post-infection. Expression of the mutant Kras and p53 was determined by RT-PCR. Both genes were expressed only in the Cre treated lines, not in GFP lines or non-transgenic lines. Sensitivity to the chemotherapeutic drug doxorubicin was also determined for the CRE, GFP or non-treated and non-transgenic fibroblast cell lines. Cell lines expressing mutant p53 and Kras were more sensitive to doxorubicin than the non-transgenic, non-treated or GFP lines. At 72 hours of treatment with 0.44 μg/ml doxorubicin the percentage of live CRE cells was approximately one third that of the other cell lines. The increased sensitivity to doxorubicin may reflect overexpression of mutant p53 and kras, relative to the wild type alleles, a behavior previously reported for tumor cells. Together, these results demonstrate that a viable, reproductively sound transgenic line of pigs has been produced in which multiple cell lineages can be transformed by cre recombination.