Development of zebrafish medulloblastoma-like PNET model by TALEN-mediated somatic gene inactivation

// Jaegal Shim 1, * , Jung-Hwa Choi 2, * , Moon-Hak Park 10 , Hyena Kim 10 , Jong Hwan Kim 3 , Seon-Young Kim 3 , Dongwan Hong 4 , Sunshin Kim 5 , Ji Eun Lee 6 , Cheol-Hee Kim 2 , Jeong-Soo Lee 7, 8, 9 and Young-Ki Bae 1, 10 1 Comparative Biomedicine Research Branch, Research Institute, National Cancer Center, Goyang, Republic of Korea 2 Department of Biology, Chungnam National University, Daejeon, Republic of Korea 3 Genome Structure Research Center, Korea Research Institute of Bioscience and Biotechnology, Daejeon, Republic of Korea 4 Clinical Genomic Analysis Branch, Research Institute, National Cancer Center, Goyang, Republic of Korea 5 Precision Medicine Branch, Research Institute, National Cancer Center, Goyang, Republic of Korea 6 Department of Health Sciences and Technology, Samsung Advanced Institute for Health Sciences and Technology, Sungkyunkwan University, Gangnam-gu, Seoul, Republic of Korea 7 Disease Target Structure Research Center, Korea Research Institute of Bioscience and Biotechnology, Daejeon, Republic of Korea 8 Convergence Research Center for Dementia DTC, Korea Institute of Science and Technology, Seoul, Republic of Korea 9 Department of Functional Genomics, Korea University of Science and Technology, Daejeon, Republic of Korea 10 Tumor Microenviroment Research Branch, Research Institute, National Cancer Center, Goyang, Republic of Korea * These authors contributed equally to this work Correspondence to: Cheol-Hee Kim, email: zebrakim@cnu.ac.kr Jeong-Soo Lee, email: jeongsoo@kribb.re.kr Young-Ki Bae, email: zebra@ncc.re.kr Keywords: medulloblastoma, PNET, MPNST, TALEN, somatic inactivation Received: November 22, 2016 Accepted: July 11, 2017 Published: July 21, 2017 ABSTRACT Genetically engineered animal tumor models have traditionally been generated by the gain of single or multiple oncogenes or the loss of tumor suppressor genes; however, the development of live animal models has been difficult given that cancer phenotypes are generally induced by somatic mutation rather than by germline genetic inactivation. In this study, we developed somatically mutated tumor models using TALEN-mediated somatic gene inactivation of cdkn2a/b or rb1 tumor suppressor genes in zebrafish. One-cell stage injection of cdkn2a/b -TALEN mRNA resulted in malignant peripheral nerve sheath tumors with high frequency (about 39%) and early onset (about 35 weeks of age) in F0 tp53 e7/e7 mutant zebrafish. Injection of rb1 -TALEN mRNA also led to the formation of brain tumors at high frequency (58%, 31 weeks of age) in F0 tp53 e7/e7 mutant zebrafish. Analysis of each tumor induced by somatic inactivation showed that the targeted genes had bi-allelic mutations. Tumors induced by rb1 somatic inactivation were characterized as medulloblastoma-like primitive neuroectodermal tumors based on incidence location, histopathological features, and immunohistochemical tests. In addition, 3′ mRNA Quanti-Seq analysis showed differential activation of genes involved in cell cycle, DNA replication, and protein synthesis; especially, genes involved in neuronal development were up-regulated.