Pancreatic cancer modeling using retrograde viral vector delivery and in vivo CRISPR/Cas9-mediated somatic genome editing.
Pancreatic cancer modeling using retrograde viral vector delivery and in vivo CRISPR/Cas9-mediated somatic genome editing.
复制标题
使用逆行病毒载体递送和体内 CRISPR/Cas9 介导的体细胞基因组编辑进行胰腺癌建模。
DOI：
10.1101/gad.264861.115
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发表时间：
2015-07-15
影响因子：
10.5
通讯作者：
Winslow MM
中科院分区：
文献类型：
--
作者：
Chiou SH;Winters IP;Wang J;Naranjo S;Dudgeon C;Tamburini FB;Brady JJ;Yang D;Grüner BM;Chuang CH;Caswell DR;Zeng H;Chu P;Kim GE;Carpizo DR;Kim SK;Winslow MM
在这项研究中，Chiou等人描述了一种通过逆行导管注射表达Cre的腺病毒和表达Cre的慢病毒载体在成人胰腺中引发可滴定肿瘤的新系统。他们还证明，CRISPR/Cas9介导的基因组编辑可以使用病毒sgRNA/Cre递送和Cre调节的Cas9小鼠等位基因在体内胰腺中有效使用，为胰腺癌中的推定肿瘤和转移抑制基因提供了一种新的系统。胰腺导管腺癌（PDAC）是一种基因组多样的，普遍的，几乎总是致命的恶性肿瘤。尽管人类PDAC的常规基因工程小鼠模型有助于理解胰腺癌的发展，但这些模型过于劳动密集，昂贵，并且进行充分了解这种疾病所需的广泛分子分析的速度缓慢。在这里，我们证明，逆行胰管注射腺病毒Cre或慢病毒Cre载体允许可滴定的胰腺肿瘤，进展为侵袭性和转移性PDAC的启动。为了在胰腺中实现体内CRISPR/Cas9介导的基因失活，我们产生了Cre调节的Cas9等位基因和表达Cre和单向导RNA的慢病毒载体。CRISPR介导的Lkb 1靶向结合致癌Kras表达导致选择失活基因组改变、Lkb 1蛋白的缺失和快速肿瘤生长，其表型模仿Cre介导的Lkb 1基因缺失。这种方法将改变我们在这种恶性肿瘤发展过程中快速询问基因功能的能力。
In this study, Chiou et al. describe a novel system for titratable tumor initiation in the adult pancreas through retrograde ductal injection of adenoviral-Cre-expressing and lentiviral-Cre-expressing vectors. They also demonstrate that CRISPR/Cas9-mediated genome editing can be used efficiently in the pancreas in vivo using viral sgRNA/Cre delivery and a Cre-regulated Cas9 mouse allele, providing a new system for putative tumor and metastatic suppressor genes in pancreatic cancer in vivo. Pancreatic ductal adenocarcinoma (PDAC) is a genomically diverse, prevalent, and almost invariably fatal malignancy. Although conventional genetically engineered mouse models of human PDAC have been instrumental in understanding pancreatic cancer development, these models are much too labor-intensive, expensive, and slow to perform the extensive molecular analyses needed to adequately understand this disease. Here we demonstrate that retrograde pancreatic ductal injection of either adenoviral-Cre or lentiviral-Cre vectors allows titratable initiation of pancreatic neoplasias that progress into invasive and metastatic PDAC. To enable in vivo CRISPR/Cas9-mediated gene inactivation in the pancreas, we generated a Cre-regulated Cas9 allele and lentiviral vectors that express Cre and a single-guide RNA. CRISPR-mediated targeting of Lkb1 in combination with oncogenic Kras expression led to selection for inactivating genomic alterations, absence of Lkb1 protein, and rapid tumor growth that phenocopied Cre-mediated genetic deletion of Lkb1. This method will transform our ability to rapidly interrogate gene function during the development of this recalcitrant cancer.