Inactivation and Inducible Oncogenic Mutation of p53 in Gene Targeted Pigs

Abstract

Mutation of the tumor suppressor p53 plays a major role in human carcinogenesis. Here we describe gene-targeted porcine mesenchymal stem cells (MSCs) and live pigs carrying a latent TP53R167H mutant allele, orthologous to oncogenic human mutant TP53R175H and mouse Trp53R172H, that can be activated by Cre recombination. MSCs carrying the latent TP53R167H mutant allele were analyzed in vitro. Homozygous cells were p53 deficient, and on continued culture exhibited more rapid proliferation, anchorage independent growth, and resistance to the apoptosis-inducing chemotherapeutic drug doxorubicin, all characteristic of cellular transformation. Cre mediated recombination activated the latent TP53R167H allele as predicted, and in homozygous cells expressed mutant p53-R167H protein at a level ten-fold greater than wild-type MSCs, consistent with the elevated levels found in human cancer cells. Gene targeted MSCs were used for nuclear transfer and fifteen viable piglets were produced carrying the latent TP53R167H mutant allele in heterozygous form. These animals will allow study of p53 deficiency and expression of mutant p53-R167H to model human germline, or spontaneous somatic p53 mutation. This work represents the first inactivation and mutation of the gatekeeper tumor suppressor gene TP53 in a non-rodent mammal.

Bone Marrow Mesenchymal Stem Cells Are an Attractive Donor Cell Type for Production of Cloned Pigs As Well As Genetically Modified Cloned Pigs by Somatic Cell Nuclear Transfer

“…Two previous studies indicated that cultured MSCs can undergo transient as well as stable genetic modification by nonviral and viral vectors (Bosch et al, 2006;Colleoni et al, 2005), but production of genetically engineered cloned pigs from MSCs was not reported in these two studies. During the preparation of this manuscript, a research group reported that they used MSCs to generate gene-targeted pig models through the SCNT technique Leuchs et al, 2012). These two studies, together with our data, clearly show that MSCs can serve as donor cells to produce genetically modified pigs by SCNT.…”

Development and translational imaging of a TP53 porcine tumorigenesis model

“…It is important to note that a model can also be too large. For example, domestic pig breeds, such as those being used in other cancer models (8)(9)(10), are 2-3 times bigger. This would exclude domestic pigs from most longitudinal monitoring/treatment studies with clinical imaging technologies, as they would quickly outgrow the size capacity of a typical clinical imaging scanner with a bore diameter of 60-70 cm.…”

“…Efforts have begun to move cancer-modeling technology previously developed in mice to new species. For example, xenograft models are being tested in immunodeficient pigs (8), and several groups, including our own in the present study, are using gene engineering technology to introduce cancer-related mutations to the porcine genome (9,10).…”

Expression of Cre Recombinase in Alveolar Epithelial Cells of the AQP2-Cre Transgenic Mini-Pigs

“…Recent advances in pig SCNT have made it possible to utilize Cre/loxP strategy in developing pig models. Leuchs S et.al described a targeted pig line carrying an inducible TP53 R167H allele orthologous to the human TP53 R175H mutation [29]. This line can be used to investigate Li Fraumeni syndrome and to decipher the oncogenic characteristics of p53 mutations found in human cancers.…”