Generation of pigs with a genetic predisposition to pancreatic cancer

• Applicants: Professorin Dr. Angelika Schnieke; Professor Dr. Eckhard Wolf
• Subject Area: Animal Breeding, Animal Nutrition, Animal Husbandry ; Evolutionary Cell and Developmental Biology (Zoology)
• Term: from 2009 to 2019
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 147416069

• Final Report Year: 2019

Final Report Abstract

The goal of the project was to generate a porcine model for pancreatic ductal adenocarcinoma model. To achieve this, a number of precisely genetically modified animals were generated, including pigs with a conditional KRAS mutation, pigs with a conditional TP53 mutation, a dual-fluorescent reporter pig for assessing Cre activity and animals designed to express Cre recombinase specifically in pancreas. All these different lines have been generated, and all are fully functional except for the Cre-line. While the lack of functional Cre pigs has been disappointing, several alternative approaches are still being investigated and we are confident that the final part of this complex undertaking will be achieved successfully. Once an effective means of delivering Cre has been determined, it can be extended to other organs and tumour types. These experiments also resulted in the establishment of additional tumour models which mimic typical human juvenile tumours (osteosarcoma, Wilm’s tumour). These are rare conditions, but nevertheless devastating and no appropriate mouse models are available. All pigs with the non-induced TP53 mutation develop osteosarcomas at an early age (6-8 month), some also develop rapidly progressing Wilm’s tumours and lymphomas. We have identified novel TP53 RNA transcripts in these pigs and are currently investigating their relevance to tumorigenesis. Similar TP53 isoforms are present in humans, where they are correlated with tumorigenesis, but interestingly are absent in the mouse. We also show that the specific p53 isoform can be used as a blood biomarker. In addition, TP73 and Hsp70 proved to be highly specific indicators of disease status. These findings are relevant to human patients and might lead to novel diagnostic or treatments. Necessary tools to make the pig a useful animal model, including organoid cultures for in vitro pathway analysis, effects of mutations and immuneinteractions have been established. Others techniques, such as immune cell histology have been been improved. The dual-fluorescent reporter pig is a novel multi-purpose resource and has been requested by several other groups to study gene delivery systems for a variety of applications.

Publications

• (2012). A porcine model of familial adenomatous polyposis. Gastroenterology 143, 1173-1175
  Flisikowska T, Merkl C, Landmann T, Eser S. Rezaei N, Cui X, Kurome M, Zakhartchenko V, Kessler B, Wieland H, Rottmann O, Schmid RM, Schneider G, Kind A, Wolf E, Saur, D, Schnieke A
  
• (2012). Inactivation and inducible oncogenic mutation of p53 in gene targeted pigs. PLoS ONE 7, e43323
  Leuchs S, Saalfrank A, Merkl C, Flisikowska T, Edlinger M, Durkovic M, Rezaei N, Kurome M, Zakhartchenko V, Kessler B, Flisikowski K, Kind A, Wolf E, Schnieke, A
  
• (2013). Fators influencing the efficiency of generating genetically engineered pigs by nuclear transfer: multifactorial analysis of a large data set. BMC Biotechnology 13(1):43
  Kurome M, Geistlinger L, Kessler B, Zakhartchenko, V, Klymiuk N, Wuensch A, Richter A, Baehr A, Kraehe K, Burkhardt K, Flisikowski K, Flisikowska T, Merkl C, Landmann M, Durkovic M, Tschukes A, Kraner, S, Schindelhauer D, Petri T. Kind A, Nagashima H, Schnieke A, Zimmer R, Wolf E
  
• (2013). Selective requirement of PI3K/PDK1 signalling for KRAS oncogene-driven pancreatic cell plasticity and cancer. Cancer Cell 23, 406-420
  Eser S., Reiff N, Messer M, Seidler B, Gottschalk K, Dobler M, Hieber H, Arbeiter A, Klein S, Kong B, Michalski CW, Schlitter AM, Esposito I, Kind AJ, Rad L, Schnieke AE, Baccarini M, Alessi DR, Rad R, Schmid RM, Schneider G, Saur D
  
• (2014) Dual fluorescent reporter pig for Cre recombination; transgene placement at the ROSA26 locus. PLoS ONE 9, e102455
  Li, S., Flisikowska, T., Kurome, M., Zakhartchenko, V., Kessler, B., Saur, D., Kind, A., Wolf, E., Flisikowski, K. and Schnieke, A.
  
• (2014). Oncogenic KRAS signalling in pancreatic cancer. Br. J. Cancer 111, 817-822
  Eser, S., Rad, R., Schnieke, A., Schneider, G. and Saur, D.
  
• (2015). Viable pigs with a conditionally-activated oncogenic KRAS mutation. Transgenic Res. 24, 509-517
  Li, S., Edlinger, M., Saalfrank, A., Flisikowski, K., Tschukes, A., Kurome, M., Zakhartchenko, V., Kessler, B., Saur, D., Kind, A., Wolf, E., Schnieke, A. and Flisikowska T.
  
• (2016). A porcine model of osteosarcoma. Oncogenesis 5, e210
  Saalfrank, A., Janssen, K-P., Ravon, M., Flisikowski, K., Eser, S., Steiger, K., Flisikowska, T., Müller-Fliedner, P., Schulze, E., Brönner, C., Gnann, A., Kappe, E., Böhm, B., Schade, B., Certa, U., Saur, D., Esposito, I., Kind, A. and Schnieke, A.
  
• (2017). Porcine familial adenomatous polyposis model enables systematic analysis of early events in adenoma progression. Scientific Reports 7, 6613
  Flisikowska, T., Stachowiak, M., Xu, H., Wagner, A., Hernandez Caceres, A., Wurmser, C., Wander, C., Pausch, H., Perkowska, A., Fischer, K., Frishman, D., Fries, R., Switonski, M., Kind, A., Saur, D., Schnieke, A. and Flisikowski, K.
  
• (2018). Genetically engineered large animals in biomedicine. In: Niemann H, Wrenzycki C (eds.) Animal Biotechnology, in 2 volumes (Reproductive biotechnologies, Emerging breeding technologies). Springer International Publishing AG, ISBN 978-3-319-92326-0 ISBN 978-3-319-92327-7
  Wolf, E., Kind, A., Aigner, B. and Schnieke, A.