Journal of Veterinary Science

The Korean Society of Veterinary Science (대한수의학회)
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In vivo multiplex gene targeting with Streptococcus pyogens and Campylobacter jejuni Cas9 for pancreatic cancer modeling in wild-type animal

  • Chang, Yoo Jin (Graduate School of International Agricultural Technology, Seoul National University) ;
  • Bae, Jihyeon (Graduate School of International Agricultural Technology, Seoul National University) ;
  • Zhao, Yang (Graduate School of International Agricultural Technology, Seoul National University) ;
  • Lee, Geonseong (Graduate School of International Agricultural Technology, Seoul National University) ;
  • Han, Jeongpil (Graduate School of International Agricultural Technology, Seoul National University) ;
  • Lee, Yoon Hoo (Graduate School of International Agricultural Technology, Seoul National University) ;
  • Koo, Ok Jae (Toolgen Inc.) ;
  • Seo, Sunmin (Department of Laboratory Animal Medicine, College of Veterinary Medicine, Konkuk University) ;
  • Choi, Yang-Kyu (Department of Laboratory Animal Medicine, College of Veterinary Medicine, Konkuk University) ;
  • Yeom, Su Cheong (Graduate School of International Agricultural Technology, Seoul National University)
  • Received : 2019.07.23
  • Accepted : 2019.12.06
  • Published : 2020.03.31
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Abstract

Pancreatic ductal adenocarcinoma is a lethal cancer type that is associated with multiple gene mutations in somatic cells. Genetically engineered mouse is hardly applicable for developing a pancreatic cancer model, and the xenograft model poses a limitation in the reflection of early stage pancreatic cancer. Thus, in vivo somatic cell gene engineering with clustered regularly interspaced short palindromic repeats is drawing increasing attention for generating an animal model of pancreatic cancer. In this study, we selected Kras, Trp53, Ink4a, Smad4, and Brca2 as target genes, and applied Campylobacter jejuni Cas9 (CjCas9) and Streptococcus pyogens Cas9 (SpCas9) for developing pancreatic cancer using adeno associated virus (AAV) transduction. After confirming multifocal and diffuse transduction of AAV2, we generated SpCas9 overexpression mice, which exhibited high double-strand DNA breakage (DSB) in target genes and pancreatic intraepithelial neoplasia (PanIN) lesions with two AAV transductions; however, wild-type (WT) mice with three AAV transductions did not develop PanIN. Furthermore, small-sized Cjcas9 was applied to WT mice with two AAV system, which, in addition, developed high extensive DSB and PanIN lesions. Histological changes and expression of cancer markers such as Ki67, cytokeratin, Mucin5a, alpha smooth muscle actin in duct and islet cells were observed. In addition, the study revealed several findings such as 1) multiple DSB potential of AAV-CjCas9, 2) peri-ductal lymphocyte infiltration, 3) multi-focal cancer marker expression, and 4) requirement of > 12 months for initiation of PanIN in AAV mediated targeting. In this study, we present a useful tool for in vivo cancer modeling that would be applicable for other disease models as well.

Keywords

Acknowledgement

This work was supported by grant from the National Research Foundation (Nos. NRF-2015R1C1A1A01051949, 2017M3A9B4061409 and 2018R1A1A1A05078158).

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