A Novel CRISPR/Cas9-mediated Mouse Model of Colon Carcinogenesis

IF 7.1 1区医学 Q1 GASTROENTEROLOGY & HEPATOLOGY

Cellular and Molecular Gastroenterology and Hepatology Pub Date : 2024-01-01 DOI:10.1016/j.jcmgh.2024.101390

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引用次数: 0

Abstract

Background & Aims

Human sporadic colorectal cancer (CRC) results from a multistep pathway with sequential acquisition of specific genetic mutations in the colorectal epithelium. Modeling CRC in vivo is critical for understanding the tumor microenvironment. To accurately recapitulate human CRC pathogenesis, mouse models must include these multi-step genetic abnormalities. The aim of this study was to generate a sporadic CRC model that more closely mimics this multi-step process and to use this model to study the role of a novel Let7 target PLAGL2 in CRC pathogenesis.

Methods

We generated a CRISPR/Cas9 somatic mutagenesis mouse model that is inducible and multiplexed for simultaneous inactivation of multiple genes involved in CRC pathogenesis. We used both a doxycycline-inducible transcriptional activator and a doxycycline-inactivated transcriptional repressor to achieve tight, non-leaky expression of the Cas9 nickase. This mouse has transgenic expression of multiple guide RNAs to induce sporadic inactivation in the gut epithelium of 4 tumor suppressor genes commonly mutated in CRC, Apc, Pten, Smad4, and Trp53. These were crossed to Vil-LCL-PLAGL2 mice, which have Cre-inducible overexpression of PLAGL2 in the gut epithelium.

Results

These mice exhibited random somatic mutations in all 4 targeted tumor suppressor genes, resulting in multiple adenomas and adenocarcinomas in the small bowel and colon. Crosses with Vil-LCL-PLAGL2 mice demonstrated that gut-specific PLAGL2 overexpression increased colon tumor growth.

Conclusions

This conditional model represents a new CRISPR/Cas9-mediated mouse model of colorectal carcinogenesis. These mice can be used to investigate the role of novel, previously uncharacterized genes in CRC, in the context of multiple commonly mutated tumor suppressor genes and thus more closely mimic human CRC pathogenesis.

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CRISPR/Cas9 介导的新型结肠癌小鼠模型。

背景与目的：人类散发性结直肠癌（CRC）是由结直肠上皮细胞中的特定基因突变依次获得的多步骤途径导致的。建立 CRC 体内模型对于了解肿瘤微环境至关重要。为了准确再现人类 CRC 的发病机制，小鼠模型必须包括这些多步骤遗传异常。目的：生成一个更接近模拟这一多步骤过程的散发性 CRC 模型，并利用该模型研究新型 Let7 靶点 PLAGL2 在 CRC 发病机制中的作用：我们建立了一个CRISPR/Cas9体细胞诱变小鼠模型，该模型具有可诱导性和多重性，可同时使多个参与CRC发病机制的基因失活。我们同时使用了强力霉素诱导的转录激活剂和强力霉素灭活的转录抑制剂，以实现 Cas9 切口酶的紧密、无漏表达。这种小鼠转基因表达了多种引导 RNA，诱导肠道上皮细胞中四个在 CRC 中常见突变的肿瘤抑制基因（Apc、Pten、Smad4 和 Trp53）零星失活。这些小鼠与Vil-LCL-PLAGL2小鼠杂交，后者在肠道上皮中Cre诱导过表达PLAGL2：结果：这些小鼠的所有四个靶向肿瘤抑制基因都出现了随机体细胞突变，导致小肠和结肠出现多发性腺瘤和腺癌。与Vil-LCL-PLAGL2小鼠杂交表明，肠道特异性PLAGL2过表达会增加结肠肿瘤的生长：这一条件模型代表了一种新的 CRISPR/Cas9 介导的结直肠癌发生小鼠模型。这些小鼠可用于在多个常见突变肿瘤抑制基因的背景下，研究以前未定性的新基因在 CRC 中的作用，从而更接近于模拟人类 CRC 的发病机制。

本文章由计算机程序翻译，如有差异，请以英文原文为准。

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来源期刊

Cellular and Molecular Gastroenterology and Hepatology Medicine-Gastroenterology

CiteScore

13.00

自引率

2.80%

发文量

246

审稿时长

42 days

期刊介绍： "Cell and Molecular Gastroenterology and Hepatology (CMGH)" is a journal dedicated to advancing the understanding of digestive biology through impactful research that spans the spectrum of normal gastrointestinal, hepatic, and pancreatic functions, as well as their pathologies. The journal's mission is to publish high-quality, hypothesis-driven studies that offer mechanistic novelty and are methodologically robust, covering a wide range of themes in gastroenterology, hepatology, and pancreatology. CMGH reports on the latest scientific advances in cell biology, immunology, physiology, microbiology, genetics, and neurobiology related to gastrointestinal, hepatobiliary, and pancreatic health and disease. The research published in CMGH is designed to address significant questions in the field, utilizing a variety of experimental approaches, including in vitro models, patient-derived tissues or cells, and animal models. This multifaceted approach enables the journal to contribute to both fundamental discoveries and their translation into clinical applications, ultimately aiming to improve patient care and treatment outcomes in digestive health.