Pancreas specific expression of oncogenes in a porcine model.

Pancreatic cancer is the fourth leading course of cancer death and early detection of the disease is crucial for successful treatment. However, pancreatic cancer is difficult to detect in its earliest stages and once symptoms appear, the cancer has often progressed beyond possibility for curing. Research into the disease has been hampered by the lack of good models. We have generated a porcine model of pancreatic cancer with use of transgenic overexpression of an oncogene cassette containing MYC, KRAS G12D and SV40 LT. The expression was initiated from a modified Pdx-1 promoter during embryogenesis in a subset of pancreatic epithelial cells. Furthermore, cells expressing the oncogenes also expressed a yellow fluorescent protein (mVenus) and an inducible negative regulator protein (rtTR-KRAB). Cells where the Pdx-1 promoter had not been activated, expressed a red fluorescent protein (Katushka). In vitro analyses of cells obtained from the transgenic pigs showed increased proliferation and expression of the transgenes when activated. Induction of the repressor protein eliminated the oncogene expression and decreased cell proliferation. In vivo analysis identified foci of pancreatic cells expressing the oncogenes at day zero post farrowing. These populations expanded and formed hyperplastic foci, with beginning abnormality at day 45. Cells in the foci expressed the oncogenic proteins and the majority of the cells were positive for the proliferation marker, Ki67. We predict that this model could be used for advanced studies in pancreatic cancer in a large animal model with focus on early detection, treatment, and identification of new biomarkers.

Multiple low-affinity interactions support binding of human osteopontin to integrin αXß2.

Integrin α(X)ß(2) (also known as complement receptor 4, p150,95, or CD11c/CD18) is expressed in the cell membrane of myeloid leukocytes. α(X)ß(2) has been reported to bind a large number of structurally unrelated ligands, often with a shared molecular character in the presence of polyanionic stretches in poorly folded proteins or glucosaminoglycans. Nevertheless, it is unclear what chemical sources of polyanionicity enable the binding by α(X)ß(2). Osteopontin (OPN) is an intrinsically disordered protein, which facilitates phagocytosis via the integrin α(X)ß(2). Unlike for other integrins, neither the RGD nor the SVVYGLR motifs account for this binding, and the molecular basis of OPN binding by α(X)ß(2) remains uncharacterized. Here, we show that the monovalent interactions between the ligand-binding domain of α(X)ß(2) and OPN, its fragments, or caseins are weak, with dissociation constants higher than 10(-5)M but with high apparent stoichiometries. From comparison with cell adhesion studies, the discrimination between α(X)ß(2) ligands and non-ligands appears to rely on these apparent stoichiometries in a way, which involves glutamate rather than aspartate side chains. Surprisingly, the extensive, negatively charged phosphorylation of OPN is not contributing to α(X)ß(2) binding. Furthermore, synchrotron radiation circular spectroscopy excludes that the phosphorylation affects the general folding of OPN. Taken together, our quantitative analyses reveal a mode of ligand recognition by integrin α(X)ß(2), which seem to differ in principles considerably from other OPN receptors.

A genetically inducible porcine model of intestinal cancer.

Transgenic porcine cancer models bring novel possibilities for research. Their physical similarities with humans enable the use of surgical procedures and treatment approaches used for patients, which facilitates clinical translation. Here, we aimed to develop an inducible oncopig model of intestinal cancer. Transgenic (TG) minipigs were generated using somatic cell nuclear transfer by handmade cloning. The pigs encode two TG cassettes: (a) an Flp recombinase-inducible oncogene cassette containing KRAS-G12D, cMYC, SV40LT - which inhibits p53 - and pRB and (b) a 4-hydroxytamoxifen (4-OHT)-inducible Flp recombinase activator cassette controlled by the intestinal epithelium-specific villin promoter. Thirteen viable transgenic minipigs were born. The ability of 4-OHT to activate the oncogene cassette was confirmed in vitro in TG colonic organoids and ex vivo in tissue biopsies obtained by colonoscopy. In order to provide proof of principle that the oncogene cassette could also successfully be activated in vivo, three pigs were perorally treated with 400 mg tamoxifen for 2 × 5 days. After two months, one pig developed a duodenal neuroendocrine carcinoma with a lymph node metastasis. Molecular analysis of the carcinoma and metastasis confirmed activation of the oncogene cassette. No tumor formation was observed in untreated TG pigs or in the remaining two treated pigs. The latter indicates that tamoxifen delivery can probably be improved. In summary, we have generated a novel inducible oncopig model of intestinal cancer, which has the ability to form metastatic disease already two months after induction. The model may be helpful in bridging the gap between basic research and clinical usage. It opens new venues for longitudinal studies of tumor development and evolution, for preclinical assessment of new anticancer regimens, for pharmacology and toxicology assessments, as well as for studies into biological mechanisms of tumor formation and metastasis.

Recombinase-Mediated Cassette Exchange (RMCE)-in Reporter Cell Lines as an Alternative to the Flp-in System.

Recombinase mediated cassette exchange (RMCE) is a powerful tool for targeted insertion of transgenes. Here we describe non-proprietary 'RMCE-in' cell lines as an alternative to the 'Flp-in' system and cell lines. RMCE-in cell lines offer a number of advantages including increased efficiency of integration of the genetic element of interest (GEI) at a single docking site, lack of bacterial backbone at the docking site both before and after GEI integration, removal of selection and visual markers initially present at the docking site upon GEI integration and the possibility to validate GEI integration by loss of a red fluorescence reporter. Moreover, the RMCE-in cell lines are compatible with GEI donors used for the Flp-in system. We demonstrate a three-step procedure for generating RMCE-in cell lines, (I) RMCE-in transposon and SB10 transposase transfection, (II) clone isolation, and (III) selecting single integrated clones with highest RFP level, which could in principle be used to turn any cell line into an RMCE-in cell line. The RMCE-in system was used as a proof of concept to produce three new RMCE-in cell lines using HEK293, HeLa, and murine embryonic stem (mES) cells. The established RMCE-in cell lines and vector are freely available from the ATCC cell bank and Addgene respectively.

Identification of transglutaminase reactive residues in human osteopontin and their role in polymerization.

Osteopontin (OPN) is a highly posttranslationally modified protein present in several tissues where it is implicated in numerous physiological processes. OPN primarily exerts its functions through interaction with integrins via the Arg-Gly-Asp and Ser-Val-Val-Tyr-Gly-Leu-Arg sequences located in the N-terminal part of the protein. OPN can be polymerized by the cross-linking enzyme transglutaminase 2 (TG2), and polymerization has been shown to enhance the biological activity of OPN. However, little is known about the reactivity and location of the glutamine and lysine residues involved in the TG2-mediated modification of OPN. Here we show that TG2 catalyses the incorporation of 5-(Biotinamido)pentylamine at glutamines in both the N- and C-terminal parts of OPN, whereas TG2 primarily incorporated the glutamine-donor peptide biotinyl-TVQQEL-OH into the C-terminal part of OPN. By mass spectrometric analyses we identified Gln34, Gln42, Gln193 and Gln248 as the major TG2 reactive glutamines in OPN. The distribution of reactive Gln and Lys residues in OPN proved to be important, as the full-length protein but not the physiologically highly active integrin-binding N-terminal part of OPN were able to polymerize in a TG2-mediated reaction. Collectively, these data provide important new molecular knowledge about the mechanism of OPN polymerization.