Abstract:
Common rodent-based models have limitations in terms of modeling human cancers. Given that pigs share many genetic and physiological similarities with humans, we investigated 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, we chose to generate transgenic pigs that can be 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 provides an 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 wound assay through which we observed a statistically significant difference of in vitro migration capability between Ad-Cre-GFP cells and Ad-GFP control cells. In a migration time of 24h, the number of cells in the wound area for the Ad-Cre-GFP cells was 184 as for the Ad-GFP cells was only 67 (p-value ≤ 0.01). A statistically significant difference was also observed between the cell cycle length for these cell lines by flow cytometry. Ad-Cre-GFP cells went through a greater number of cell divisions compared with Ad-GFP cells. Within a 73h time period, Ad-Cre-GFP cells divided twice as many times than Ad-GFP cells (p-value ≤ 0.01). Present results demonstrate that induction of the transgenes in these porcine cells triggered a transformed phenotype. In the future, these cells will be tested for growth in soft agar, tumor growth in mice, and then the pigs will be monitored for tumor incidence following site-specific transgene induction. Such an approach could provide a porcine model to cancer etiology and the development of anti-cancer therapy.
Research also presented at the 59th Brazilian Congress of Genetics, September 16-19, 2013, Sao Paulo, Brazil.