A pipeline for rapidly generating genetically engineered mouse models of pancreatic cancer using in vivo CRISPRCas9 mediated somatic recombination

ABSTRACTGenetically engineered mouse models (GEMMs) that recapitulate the major genetic drivers in pancreatic ductal adenocarcinoma (PDAC) have provided unprecedented insights into the pathogenesis of this lethal neoplasm. Nonetheless, generating an autochthonous model is an expensive, time consuming and labor intensive process, particularly when tissue specific expression or deletion of compound alleles are involved. In addition, many of the current PDAC GEMMs cause embryonic, pancreas-wide activation or loss of driver alleles, neither of which reflects the cognate human disease scenario. The advent of CRISPR/Cas9 based gene editing can potentially circumvent many of the aforementioned shortcomings of conventional breeding schema, but ensuring the efficiency of gene editing in vivo remains a challenge. Here we have developed a pipeline for generating PDAC GEMMs of complex genotypes with high efficiency using a single “workhorse” mouse strain expressing Cas9 in the adult pancreas under a p48 promoter. Using adeno-associated virus (AAV) mediated delivery of multiplexed guide RNAs (sgRNAs) to the adult murine pancreas of p48-Cre; LSL-Cas9 mice, we confirm our ability to express an oncogenic KrasG12D allele through homology-directed repair (HDR), in conjunction with CRISPR-induced disruption of cooperating alleles (Trp53, Lkb1 and Arid1A). The resulting GEMMs demonstrate a spectrum of precursor lesions (pancreatic intraepithelial neoplasia [PanIN] or Intraductal papillary mucinous neoplasm [IPMN] with eventual progression to PDAC. Next generation sequencing of the resulting murine PDAC confirms HDR of oncogenic KrasG12D allele at the endogenous locus, and insertion deletion (“indel”) and frameshift mutations of targeted tumor suppressor alleles. By using a single “workhorse” mouse strain and optimal AAV serotype for in vivo gene editing with combination of driver alleles, we have created a facile autochthonous platform for interrogation of the PDAC genome.

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Pancreatic Intraepithelial Neoplasia and Pancreatic Tumorigenesis: Of Mice and Men

Archives of Pathology & Laboratory Medicine ◽

10.5858/133.3.375 ◽

2009 ◽

Vol 133 (3) ◽

pp. 375-381 ◽

Cited By ~ 7

Author(s):

Niki A. Ottenhof ◽

Anya N. A. Milne ◽

Folkert H. M. Morsink ◽

Paul Drillenburg ◽

Fiebo J. W. ten Kate ◽

...

Keyword(s):

Pancreatic Cancer ◽

Tumor Progression ◽

Mouse Models ◽

Intraepithelial Neoplasia ◽

Genetic Alterations ◽

Genetically Engineered ◽

Ductal Adenocarcinoma ◽

Pancreatic Intraepithelial Neoplasia ◽

Pancreatic Carcinogenesis ◽

Genetically Engineered Mouse Models

Abstract Context.—Pancreatic cancer has a poor prognosis with a 5-year survival of less than 5%. Early detection is at present the only way to improve this outlook. This review focuses on the recent advances in our understanding of pancreatic carcinogenesis, the scientific evidence for a multistaged tumor progression, and the role genetically engineered mouse models can play in recapitulating the natural course and biology of human disease. Objectives.—To illustrate the stepwise tumor progression of pancreatic cancer and genetic alterations within the different stages of progression and to review the findings made with genetically engineered mouse models concerning pancreatic carcinogenesis. Data Sources.—A review of recent literature on pancreatic tumorigenesis and genetically engineered mouse models. Conclusions.—Pancreatic cancer develops through stepwise tumor progression in which preinvasive stages, called pancreatic intraepithelial neoplasia, precede invasive pancreatic cancer. Genetic alterations in oncogenes and tumor suppressor genes underlying pancreatic cancer are also found in pancreatic intraepithelial neoplasia. These mutations accumulate during progression through the consecutive stages of pancreatic intraepithelial neoplasia lesions. Also in genetically engineered mouse models of pancreatic ductal adenocarcinoma, tumorigenesis occurs through stepwise progression via consecutive mouse pancreatic intraepithelial neoplasia, and these models provide important tools for clinical applications. Nevertheless differences between mice and men still remain.

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Development of multi-drug loaded PEGylated nanodiamonds to inhibit tumor growth and metastasis in genetically engineered mouse models of pancreatic cancer

Nanoscale ◽

10.1039/c9nr05478b ◽

2019 ◽

Vol 11 (45) ◽

pp. 22006-22018 ◽

Cited By ~ 4

Author(s):

Vijay Sagar Madamsetty ◽

Krishnendu Pal ◽

Sandeep Keshavan ◽

Thomas R. Caulfield ◽

Shamit Kumar Dutta ◽

...

Keyword(s):

Pancreatic Cancer ◽

Mouse Models ◽

Genetically Engineered ◽

Inhibit Tumor Growth ◽

Schematic Representation ◽

Genetically Engineered Mouse Models ◽

Tumor Growth And Metastasis ◽

Nano Formulation ◽

Immune Competent Mouse

Schematic representation demonstrating the fabrication and in vivo evaluation of an immune-modulatory nano-formulation consisting of irinotecan and curcumin in immune-competent mouse models of pancreatic adenocarcinoma.

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How Genetically Engineered Mouse Tumor Models Provide Insights Into Human Cancers

Journal of Clinical Oncology ◽

10.1200/jco.2010.30.8304 ◽

2011 ◽

Vol 29 (16) ◽

pp. 2273-2281 ◽

Cited By ~ 72

Author(s):

Katerina Politi ◽

William Pao

Keyword(s):

Mouse Models ◽

Cancer Biology ◽

Human Cancer ◽

Genetically Engineered ◽

Mouse Tumor ◽

Tumor Models ◽

Genetically Engineered Mouse Models ◽

Human Cancers ◽

Chemically Induced

Genetically engineered mouse models (GEMMs) of human cancer were first created nearly 30 years ago. These early transgenic models demonstrated that mouse cells could be transformed in vivo by expression of an oncogene. A new field emerged, dedicated to generating and using mouse models of human cancer to address a wide variety of questions in cancer biology. The aim of this review is to highlight the contributions of mouse models to the diagnosis and treatment of human cancers. Because of the breadth of the topic, we have selected representative examples of how GEMMs are clinically relevant rather than provided an exhaustive list of experiments. Today, as detailed here, sophisticated mouse models are being created to study many aspects of cancer biology, including but not limited to mechanisms of sensitivity and resistance to drug treatment, oncogene cooperation, early detection, and metastasis. Alternatives to GEMMs, such as chemically induced or spontaneous tumor models, are not discussed in this review.

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Illuminating Cancer Systems with Genetically Engineered Mouse Models and Coupled Luciferase Reporters In Vivo

Cancer Discovery ◽

10.1158/2159-8290.cd-12-0503 ◽

2013 ◽

Vol 3 (6) ◽

pp. 616-629 ◽

Cited By ~ 39

Author(s):

Brandon Kocher ◽

David Piwnica-Worms

Keyword(s):

Mouse Models ◽

Genetically Engineered ◽

Genetically Engineered Mouse Models ◽

Luciferase Reporters

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Genetic Mutations of Pancreatic Cancer and Genetically Engineered Mouse Models

Cancers ◽

10.3390/cancers14010071 ◽

2021 ◽

Vol 14 (1) ◽

pp. 71

Author(s):

Yuriko Saiki ◽

Can Jiang ◽

Masaki Ohmuraya ◽

Toru Furukawa

Keyword(s):

Pancreatic Cancer ◽

Mouse Models ◽

Tumor Biology ◽

Genetically Engineered ◽

Lineage Tracing ◽

Ductal Adenocarcinoma ◽

Pancreatic Tumors ◽

Precursor Lesions ◽

Genetically Engineered Mouse Models ◽

Molecular Alterations

Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive malignancy, and the seventh leading cause of cancer-related deaths worldwide. An improved understanding of tumor biology and novel therapeutic discoveries are needed to improve overall survival. Recent multi-gene analysis approaches such as next-generation sequencing have provided useful information on the molecular characterization of pancreatic tumors. Different types of pancreatic cancer and precursor lesions are characterized by specific molecular alterations. Genetically engineered mouse models (GEMMs) of PDAC are useful to understand the roles of altered genes. Most GEMMs are driven by oncogenic Kras, and can recapitulate the histological and molecular hallmarks of human PDAC and comparable precursor lesions. Advanced GEMMs permit the temporally and spatially controlled manipulation of multiple target genes using a dual-recombinase system or CRISPR/Cas9 gene editing. GEMMs that express fluorescent proteins allow cell lineage tracing to follow tumor growth and metastasis to understand the contribution of different cell types in cancer progression. GEMMs are widely used for therapeutic optimization. In this review, we summarize the main molecular alterations found in pancreatic neoplasms, developed GEMMs, and the contribution of GEMMs to the current understanding of PDAC pathobiology. Furthermore, we attempted to modify the categorization of altered driver genes according to the most updated findings.

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Genetically Engineered Mouse Models of Liver Tumorigenesis Reveal a Wide Histological Spectrum of Neoplastic and Non-Neoplastic Liver Lesions

Cancers ◽

10.3390/cancers12082265 ◽

2020 ◽

Vol 12 (8) ◽

pp. 2265

Author(s):

Katja Steiger ◽

Nina Gross ◽

Sebastian A. Widholz ◽

Roland Rad ◽

Wilko Weichert ◽

...

Keyword(s):

Mouse Models ◽

Liver Tumors ◽

Genetically Engineered ◽

Liver Lesions ◽

Genetically Engineered Mouse Models ◽

Chemically Induced ◽

Wide Range ◽

Correct Application ◽

Huge Variety

Genetically engineered mouse models (GEMM) are an elegant tool to study liver carcinogenesis in vivo. Newly designed mouse models need detailed (histopathological) phenotyping when described for the first time to avoid misinterpretation and misconclusions. Many chemically induced models for hepatocarcinogenesis comprise a huge variety of histologically benign and malignant neoplastic, as well as non-neoplastic, lesions. Such comprehensive categorization data for GEMM are still missing. In this study, 874 microscopically categorized liver lesions from 369 macroscopically detected liver “tumors” from five different GEMM for liver tumorigenesis were included. The histologic spectrum of diagnosis included a wide range of both benign and malignant neoplastic (approx. 82%) and non-neoplastic (approx. 18%) lesions including hyperplasia, reactive bile duct changes or oval cell proliferations with huge variations among the various models and genetic backgrounds. Our study therefore critically demonstrates that models of liver tumorigenesis can harbor a huge variety of histopathologically distinct diagnosis and, depending on the genotype, notable variations are expectable. These findings are extremely important to warrant the correct application of GEMM in liver cancer research and clearly emphasize the role of basic histopathology as still being a crucial tool in modern biomedical research.

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Cell Type of Pancreatic Ductal Adenocarcinoma Origin: Implications for Prognosis and Clinical Outcomes

Visceral Medicine ◽

10.1159/000520946 ◽

2021 ◽

pp. 1-6

Author(s):

Shilpa Patil ◽

Yan Dou ◽

Janel L. Kopp

Keyword(s):

Pancreatic Ductal Adenocarcinoma ◽

Mouse Models ◽

Pancreatic Injury ◽

Genetically Engineered ◽

Ductal Adenocarcinoma ◽

Cell Of Origin ◽

Cell Type ◽

Genetically Engineered Mouse Models ◽

Cellular Origin ◽

Ductal Cells

Background: Pancreatic ductal adenocarcinoma (PDAC) is a devastating disease that has no effective early detection method or treatment to date. Summary: The normal cell type that initiates PDAC, or its cellular origin, is still unknown. To investigate the contribution of distinct normal epithelial cell types to PDAC tumorigenesis, genetically engineered mouse models were used to show that both acinar and ductal cells are capable of giving rise to PDAC. These studies indicated that genetic mutations and pancreatic injury interact differently with each cellular origin to affect their predilection and process for forming PDAC. In this review, we summarize recent findings using various genetically engineered mouse models in the identification and characterization of the PDAC cell of origin. We also discuss potential implications for cellular origin on tumor development, PDAC transcriptional subtype, and disease prognosis of patients. Key Message: Although it is clear that both ductal and acinar cells have the potential to form PDAC, whether cellular origin can indeed influence patient prognosis and whether knowledge of cellular origin will aid in the diagnosis or treatment of patients in the future will need further study.

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Deciphering the RAS/ERK pathway in vivo

Biochemical Society Transactions ◽

10.1042/bst20160135 ◽

2017 ◽

Vol 45 (1) ◽

pp. 27-36 ◽

Cited By ~ 21

Author(s):

Coralie Dorard ◽

Georg Vucak ◽

Manuela Baccarini

Keyword(s):

Mouse Models ◽

Short Review ◽

Genetically Engineered ◽

Tissue Homeostasis ◽

Pharmacological Intervention ◽

Erk Pathway ◽

Erk Activation ◽

Genetically Engineered Mouse Models ◽

Mutational Activation

The RAS/ERK pathway has been intensely studied for about three decades, not least because of its role in human pathologies. ERK activation is observed in the majority of human cancers; in about one-third of them, it is driven by mutational activation of pathway components. The pathway is arguably one of the best targets for molecule-based pharmacological intervention, and several small-molecule inhibitors are in clinical use. Genetically engineered mouse models have greatly contributed to our understanding of signaling pathways in development, tissue homeostasis, and disease. In the specific case of the RAS/ERK pathway, they have revealed unique biological roles of structurally and functionally similar proteins, new kinase-independent effectors, and unsuspected relationships with other cascades. This short review summarizes the contribution of mouse models to our current understanding of the pathway.

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The Use of Genetically Engineered Mouse Models for Studying the Function of Mutated Driver Genes in Pancreatic Cancer

Journal of Clinical Medicine ◽

10.3390/jcm8091369 ◽

2019 ◽

Vol 8 (9) ◽

pp. 1369 ◽

Cited By ~ 2

Author(s):

Weng ◽

Lin ◽

Cheng

Keyword(s):

Pancreatic Cancer ◽

Mouse Models ◽

Cancer Biology ◽

Gene Knockout ◽

Genetically Engineered ◽

Ductal Adenocarcinoma ◽

Neoplastic Progression ◽

Driver Genes ◽

Genetically Engineered Mouse Models ◽

Oncology Research

Pancreatic cancer is often treatment-resistant, with the emerging standard of care, gemcitabine, affording only a few months of incrementally-deteriorating survival. Reflecting on the history of failed clinical trials, genetically engineered mouse models (GEMMs) in oncology research provides the inspiration to discover new treatments for pancreatic cancer that come from better knowledge of pathogenesis mechanisms, not only of the derangements in and consequently acquired capabilities of the cancer cells, but also in the aberrant microenvironment that becomes established to support, sustain, and enhance neoplastic progression. On the other hand, the existing mutational profile of pancreatic cancer guides our understanding of the disease, but leaves many important questions of pancreatic cancer biology unanswered. Over the past decade, a series of transgenic and gene knockout mouse modes have been produced that develop pancreatic cancers with features reflective of metastatic pancreatic ductal adenocarcinoma (PDAC) in humans. Animal models of PDAC are likely to be essential to understanding the genetics and biology of the disease and may provide the foundation for advances in early diagnosis and treatment.

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