An inducible large animal cancer model

L.B. Schook
Swine in Biomedical Research Meeting, July 6-8, 2014, Raleigh, NC


Common rodent-based models have limitations in terms of modeling human cancers. Given that pigs share many genetic and physiological similarities with humans, we investigate the potential of developing genetic porcine models of cancer.  In this regard, we previously reported that activation of oncogenes like Ras in conjunction with inhibiting tumor suppressor pathways like p53 were required, in part, to convert normal porcine cells to a tumorigenic state. Based on this, transgenic pigs were generated that could induced to express oncogenic Kras and dominant-negative p53. First, porcine Kras and p53 wild-type genes were cloned, sequenced and aligned with porcine, human and murine homologues to identify porcine-specific mutation sites corresponding to those commonly found in human cancers. Porcine Kras mutation occurs at the 12th glycine (G) to aspartic acid (D), whereas p53 arginine (R) at 167th position was mutated to histidine (H). KrasG12D and p53R167H mutants were linked by internal ribosome entry sites (IRES) for their simultaneous expression and then inserted into a vector following the LoxP-polyA(STOP)-LoxP sequence (LSL). Porcine fetal fibroblasts were transfected in vitro with this vector construct and infected by adenovirus (Ad) vectors encoding Cre recombinase (Ad-Cre-GFP), which deletes the LSL sequence and permits transgene expression, or control Ad vectors without the inserted Cre transgene (Ad-GFP). Cre recombinase-mediated KrasG12D and p53R167H expression was significantly induced in porcine fibroblasts transfected with Ad-Cre-GFP virus compared with Ad-GFP control, which provided in vitro proof of functional test of the “oncopig” construct. We then transfected porcine fibroblasts with the aforementioned “oncopig” construct to produce donor cell lines for nuclear transfer cloning.  Transgenic fibroblast cell lines generated from the cloned pig were subjected to a battery of tests to demonstrate a transformed phenotype including tumor formation in SCID mice. Present results demonstrate that tumors can be induced by Ade-Cre injection into transgenic pigs.  This approach provides a porcine model for early cancer detection and the development of anti-cancer therapies.