Chang Yoo Jin, Bae Jihyeon, Zhao Yang, Lee Geonseong, Han Jeongpil, Lee Yoon Hoo, Koo Ok Jae, Seo Sunmin, Choi Yang Kyu, Yeom Su Cheong
Graduate School of International Agricultural Technology, Seoul National University, Pyeongchang 25354, Korea.
Department of Pharmacology, Yonsei University College of Medicine, Seoul 03722, Korea.
J Vet Sci. 2020 Mar;21(2):e26. doi: 10.4142/jvs.2020.21.e26.
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, 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 , , , , and as target genes, and applied 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.
胰腺导管腺癌是一种致命的癌症类型，与体细胞中的多个基因突变相关。基因工程小鼠很难用于建立胰腺癌模型，而异种移植模型在反映早期胰腺癌方面存在局限性。因此，利用成簇规律间隔短回文重复序列进行体细胞基因工程在构建胰腺癌动物模型方面越来越受到关注。在本研究中，我们选择了 、 、 、 和 作为靶基因，并应用嗜热栖热菌Cas9（CjCas9）和化脓性链球菌Cas9（SpCas9），通过腺相关病毒（AAV）转导来构建胰腺癌模型。在确认AAV2的多灶性和弥漫性转导后，我们构建了SpCas9过表达小鼠，经两次AAV转导后，这些小鼠在靶基因中表现出高度的双链DNA断裂（DSB）以及胰腺上皮内瘤变（PanIN）病变；然而，经三次AAV转导的野生型（WT）小鼠并未发生PanIN。此外，将小型Cjcas9应用于具有双AAV系统的WT小鼠，这些小鼠还出现了高度广泛的DSB和PanIN病变。观察了导管和胰岛细胞中的组织学变化以及癌症标志物如Ki67、细胞角蛋白、粘蛋白5a、α平滑肌肌动蛋白的表达。此外，该研究还揭示了一些发现，如1）AAV - CjCas9的多重DSB潜力，2）导管周围淋巴细胞浸润，3）多灶性癌症标志物表达，以及4）在AAV介导的靶向中启动PanIN需要超过12个月。在本研究中，我们提出了一种用于体内癌症建模的有用工具，该工具也适用于其他疾病模型。
