Name: a genetically engineered mouse model of sporadic colorectal cancer
Uploaded: 2017-07-06T12:30:00.000+00:00
Duration: 6 min 1 s
Description: Watch this Scientific Journal Video about A Genetically Engineered Mouse Model of Sporadic Colorectal Cancer at JoVE.com

这项工作致力于莫里茨·科赫教授的记忆。

这里提出的动物实验由机构和政府动物护理和使用委员会进行独立审查和批准，并根据实验动物科学协会联合会（FELASA）指导进行。采取一切可能的措施，尽量减少包括麻醉和镇痛在内的痛苦，或必要时提早安乐死。 局部肿瘤感染通过外科手术感染<ol><li> 手术动物的准备 注意:几乎任何条件（"floxed"）突变可以通过这里描述的方法诱导。推荐在大肠癌如Apc，Kras或Tp53相关基因中使用突变。 cre重组的效率取决于要切除的构建体的大小。大量的花粉序列的排除效率较低。所有等位基因的重组应通过PC在肿瘤中得到证实R. <ol><li>对于结肠直肠肿瘤的发展，使用以下条件等位基因的交叉进行CRC的基本模型（括号中的MGI数据库号）: Apc tm2Rak （MGI:3688435） 16 Kras tm4Tyj （MGI:2429948） 17 Tp53 tm2Tyj （MGI:3039263） 18 </li><li>如果需要荧光报告基因等位基因（ 例如 ，检测微转移），请使用以下等位基因: Gt（ROSA）26Sor tm6（CAG-ZsGreen1）Hze （MGI:3809522） 19 注意:所有上述菌株均可通过NCI Mouse Repository或Jackson Laboratory获得。不需要空腹，因为所有剩余的粪便可以在腺病毒感染之前被冲洗掉。术前禁食导致较高的围手术期死亡率，并对小型啮齿动物造成巨大压力。 </li><li>使用3〜3.5体积％的七氟烷进行全身麻醉。亏损t他的脚趾反射指示足够的麻醉。 </li><li>在第一次切开之前，皮下注射0.05 mg / kg的丁丙诺啡。 </li><li>用眼科软膏覆盖麻醉小鼠的眼睛，以避免角膜干燥。 </li><li>将小鼠放在小桌子的仰卧位置。使用非创伤性胶带来限制鼠标。 </li><li>用电动剃须刀剃除腹部（脱毛膏可以替代使用），并用酒精拭子或碘进行消毒。请使用制造商推荐的接触时间。 </li><li>用无菌盖子覆盖手术区域。 注意:围手术期抗生素的使用是可选的，并遵守机构准则。 </li><li>使用无菌单次使用或无菌仪器进行所有外科手术。 </li></ol></li><li> 中线剖腹术和结肠暴露 <ol><li>使用剪刀（可以替代使用手术刀）进行中线切口（〜15毫米）的下腹部皮肤。 </li><li>用镊子拿起腹壁肌肉组织，用剪刀仔细切开，打开腹腔。 </li><li>识别远端结肠，只用无创伤镊子触摸。用肛门近端约15毫米的精密夹具（ 例如 ，Micro Serrefine血管夹）夹住结肠。 注意:随时要特别注意冒号的脆弱性。穿孔不可避免地导致腹膜炎和败血症，需要动物的安乐死。 </li></ol></li><li> 用Adeno-cre病毒分段结肠感染 <ol><li>插入灵活的特氟隆管，经过狭窄并小心推进，直至达到预先放置在距离肛门边15mm处的夹具达到的管腔闭塞。不要使用过量的力，因为这可能会导致穿孔。 </li><li>用30G套管插管，连接标准的1 mL注射器并冲洗colon用生理盐水排出剩余的粪便物质。这可能需要几mL盐水。 </li><li>一旦远端结肠清空，就取出管子并用新鲜的特氟龙管替代，并将其直接如上所述位于夹具远端。 </li><li>使用第二个夹子将结肠置于距离近端夹具远端3 mm处（ 即 ，插入管中，距离肛门边缘约12 mm），导致3 mm的待分离的段被感染。 注意:对于远端闭塞部分，Fogarty冠状动脉夹片已被证明是最适合的，因为它们是橡胶化的，导致结肠紧闭，尽管夹子分支之间的管腔内管。 </li><li>使用第二个注射器（标准1 mL，30G插管）小心地将50 - 80μL的0.25％胰蛋白酶-EDTA注入到夹住的结肠段，并孵育10分钟。将插管和注射器连接到特氟龙管上，以防止流体泄漏。 <li>首先取下远端夹具，然后取出胰蛋白酶管。 </li><li>用约500μL生理盐水冲洗远端结肠，以除去剩余的胰蛋白酶。 </li><li>插入新的特氟隆管，将远端夹钳放回原位，并用50 - 80μL腺病毒溶液（10 11噬菌斑形成单位（PFU）/ mL，在磷酸盐缓冲盐水中）充满结肠段，并孵育30分钟图2A ）。 注意:不要溢出病毒溶液，因为与腺苷接触可能会导致有条件突变小鼠的任何组织中的肿瘤发展。 </li><li>拆下夹子和管子。 </li></ol></li><li> 腹部闭合和术后恢复 <ol><li>关闭腹壁6-0快速吸收跑步su（ 例如 ，聚二恶烷酮（PDS））。 </li><li>用手术伤口夹闭合皮肤。 </li><li>将鼠标置于38°C的加热垫上，直至从麻醉完全恢复。 </li><li>在手术后12小时再次施用0.05 mg / kg丁丙诺啡，然后每隔12 h再次使用丁丙诺啡，如有需要， </li><li>每天至少监测小鼠一次，因肿瘤生长引起的痛苦征兆。 </li></ol></li></ol>结肠镜检查注意:根据所使用的条件突变，腺病毒感染在2-4周内导致内窥镜可见的肿瘤。因此，在腺病毒诱导后2周进行第一次术后结肠镜检查，每2周重复一次。推荐用于小肠结肠镜检查的市售系统。 20 <ol><li> 用于结肠镜检查的动物的制备 注意:不需要空腹编辑。剩余的粪便通常在远端结肠中形成良好，并且可以在结肠镜检查期间被推出超过肿瘤，从而使不必要的反复禁食的紧张过程。 <ol><li>使用3〜3.5体积％的七氟烷进行全身麻醉。脚趾反射的丧失表示足够的麻醉。 </li><li>用眼科软膏覆盖麻醉小鼠的眼睛，以避免角膜干燥。 </li><li>将小鼠在仰卧位上放在小桌子上。 </li></ol></li><li> 结肠镜检查 <ol><li>将范围（直径1.9毫米，长度10厘米）通过肛门插入肠道，并在视觉控制下小心地吹入空气以扩大结肠。不要比检查要求更多的空气。 注意:对于空气吹入，可以使用结肠镜检查系统的防雾气泵。如果没有防雾气泵可用，则可以使用任何其他具有非常低压力设置的气泵柔软的空气与精致的减压阀。二氧化碳（CO 2 ）容易导致小型啮齿动物酸中毒，因此必须避免。 </li><li>仔细推动范围，直到远端结肠的粘膜病变可以被识别（ 图1A-1D ）。 </li><li>保存内窥镜图像供以后评估。之前已经描述了用于管腔内肿瘤的内窥镜评分系统。 20 </li><li>仔细取出范围，将鼠标置于38°C的加热垫上，直到其从麻醉中完全恢复。 </li></ol></li></ol>

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作者没有什么可以披露的。

虽然它们通常易于产生和维持，但基于细胞系注射的经典CRC小鼠模型是人为的，并且不能完全重现人类疾病。因此，GEMM已经开发。第一个CRC GEMM是Apc Min小鼠，其在Apc基因中携带杂合的无效突变，因此模拟人类遗传性家族性腺瘤性息肉病（FAP）。然而，Apc Min小鼠总是发展出不限于结肠的多个肠腺瘤;此外，腺瘤形成的时间和确切位置是随机的，并且当腺瘤可以进展之前，随着小鼠死...

结肠直肠癌（CRC）仍然是西方国家与癌症相关死亡的主要原因之一。 1尽管早期疾病患者的预后良好，但是在后期阶段诊断出许多肿瘤，尽管有许多治疗方案，预后有限。 2，3，4，5 目前大多数CRC的小鼠模型都是基于将衍生自细胞系或患者肿瘤的肿瘤细胞植入免疫缺陷小鼠。 6,7,8这导致局部，并且取决于注射部位和用于注射的肿瘤细胞，有时是转移性肿瘤。 9，10然而，所得到的异种移植模型具有巨大的作用r限制。它们必须建立在免疫缺陷小鼠中，从而消除肿瘤与宿主免疫系统之间的复杂相互作用。另外，由于肿瘤基质来自宿主细胞，所以人类肿瘤实质与鼠基质之间的相互作用是有缺陷的，因此不能代表疾病。这些缺陷可以通过使用注射用鼠细胞系来避免。然而，只有少数鼠CRC细胞系可用，并且与大多数可用的人类CRC细胞系相似，是单克隆和高度间变性的。总之，目前大多数目前可用的CRC小鼠模型是高度人为的，并不完全代表人类疾病。 CRC的遗传工程小鼠模型（GEMM）可以避免这些缺点，因为它们具有通过诱导结肠中CRC的关键突变而产生的真正的小鼠肿瘤。 12，13 ，这可以通过在结肠直肠粘膜中通过cre重组酶激活条件（floxed）种系突变来实现。在许多其他肿瘤实体的GEMM中，使用由组织特异性启动子驱动的种系（诱导型）cre表达，种系cre不能用于结肠，因为这导致整个结肠中的大量腺瘤通过良性肿瘤负荷导致死亡很年轻因此，在这里描述的模型中，使用表达cre的腺病毒载体来感染短的结肠段。这导致在研究者定义的时间点诱导该粘膜部分的肿瘤发生，导致腺瘤最终进展到侵袭性和转移性癌。肿瘤是真正的小鼠肿瘤，在完整的微环境中生长，因此能够模拟包括肿瘤 - 宿主相互作用和转移性级联在内的整体结直肠癌发生。这个模型是因此是癌症生物学和临床前治疗试验研究的有吸引力的平台。 CRC遗传工程小鼠模型的一个主要缺点是其技术复杂性。以前已经描述了使用直肠腺苷灌注剂的小鼠携带Floced Apc等位基因的局部妊娠然而，这种技术，肠肿瘤的发生率，多样性和位置可能是高度可变的。因此，开发了通过手术夹紧待诱导的片段限制腺病毒感染的技术。 13我们修改了这一程序，以改善动物福利，并减少死亡率和肿瘤数量。有了这个协议，所有具有小型啮齿动物手术经验的实验室都应该能够复制模型并产生高度可重复性且容易接近结肠镜检查的肿瘤。取决于条件m用于肿瘤发生的术语，腺瘤的全谱，侵袭性癌和转移灶可以观察到。由于肿瘤位于远端结肠，因此在该模型中可以很容易进行连续内镜评估。

<table><tbody><tr><td>&lt;strong&gt;Reagents / consumables&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Dulbecco&#039;s Phosphate Buffered Saline</td><td>Life Technologies GmbH</td><td>14190169</td><td></td></tr><tr><td>Trypsin-EDTA (0.25%, Phenol-Red)</td><td>Life Technologies GmbH</td><td>25200072</td><td></td></tr><tr><td>Normal saline 0.9% (E154)</td><td>Serumwerk Bernburg AG</td><td>10013</td><td></td></tr><tr><td>Aqua ad injectabilia</td><td>B. Braun Melsungen AG</td><td>235144</td><td></td></tr><tr><td>Ad5CMV-Cre (adenovirus, c = 2E+11 PFU/mL)</td><td>Gene Transfer Vector Core&lt;br/&gt; University of Iowa</td><td></td><td></td></tr><tr><td>15 mL, 50 mL centrifuge tubes</td><td>Greiner Bio-One GmbH</td><td>188271/227270</td><td></td></tr><tr><td>Eppendorf tubes 1.5 mL/ 2 mL</td><td>Sarstedt AG &amp;amp; Co.</td><td>72,695,400</td><td></td></tr><tr><td>Petri dish PS 100/15 mm (sterile, Nuclon)</td><td>Fisher Scientific GmbH</td><td>10508921/ NUNC150350</td><td></td></tr><tr><td>1 mL Syringe (without dead volume) - Injekt-F SOLO</td><td>Braun/neoLab</td><td>194291661</td><td></td></tr><tr><td>30G injection needle</td><td>BECTON DICKINSON</td><td>304000</td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Analgesia / anesthesia&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Sevoflurane (Sevoflurane AbbVie)</td><td>AbbVie Germany GmbH &amp;amp; Co. KG</td><td>-</td><td></td></tr><tr><td>Medical oxygen</td><td>Air Liquide Medical GmbH</td><td>-</td><td></td></tr><tr><td>Buprenorphine (Temgesic)</td><td>Indivior Eu Ltd.</td><td>-</td><td></td></tr><tr><td>Bepanthen - ophthalmic ointment</td><td>Bayer Vital GmbH</td><td>10047757</td><td></td></tr><tr><td>Table Top Research Anesthesia Machine x/O2 Flush w/ Sevoflurane Vaporizer</td><td>Parkland Scientific</td><td>V3000PS/PK</td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Surgical Equipment&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Cellulose swabs</td><td>Lohmann &amp;amp; Rauscher Deutschland</td><td>13356</td><td></td></tr><tr><td>Insulin syringe EMG 1 mL (with 30G cannula)</td><td>B. Braun Melsungen AG</td><td>9161627S</td><td></td></tr><tr><td>Fine Bore Tubing (bore: 0.28 mm/ diameter: 0.61mm)</td><td>Smiths Medical Deutschland</td><td>800/100/100</td><td></td></tr><tr><td>Micro-Adson Forceps</td><td>Fine Science Tools</td><td>11018-12</td><td></td></tr><tr><td>Iris Scissor - ToughCut</td><td>Fine Science Tools</td><td>14058-11</td><td></td></tr><tr><td>Olsen-Hegar Needle Holder</td><td>Fine Science Tools</td><td>12002-12</td><td></td></tr><tr><td>AutoClip Kit</td><td>Fine Science Tools</td><td>12020-00</td><td></td></tr><tr><td>PDS Z1012H 6/0 C1 (surgical suture)</td><td>Johnson &amp;amp; Johnson Medical GmbH</td><td>Z1012H</td><td></td></tr><tr><td>Curved Micro Serrefine Vascular Clamp</td><td>Fine Science Tools</td><td>18055-05</td><td></td></tr><tr><td>Fogarty Spring Clips</td><td>Edwards</td><td>CDSAFE 6</td><td></td></tr><tr><td>Hot Plate 062</td><td>Labotect</td><td>13854</td><td></td></tr><tr><td>Isis - Hair shaver</td><td>Aesculap - Braun</td><td>-</td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Colonoscopy&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Cold Light Fountain XENON 175 SCB</td><td>Karl Storz</td><td>20132101-1</td><td>Karl Storz Coloview System Mainz</td></tr><tr><td>Fiber Optic Light Cable</td><td>Karl Storz</td><td>69495NL</td><td>Karl Storz Coloview System Mainz</td></tr><tr><td>TRICAM Three-Chip Camera Head</td><td>Karl Storz</td><td>20221030</td><td>Karl Storz Coloview System Mainz</td></tr><tr><td>TRICAM SLII Camera Control Unit</td><td>Karl Storz</td><td>20223011-1</td><td>Karl Storz Coloview System Mainz</td></tr><tr><td>15&quot; Flat Screen Monitor EndoVue</td><td>Karl Storz</td><td>9415NN</td><td>Karl Storz Coloview System Mainz</td></tr><tr><td>HOPKINS Straight Forward Telescope&lt;br/&gt; diameter 1.9 mm; length 10 cm&lt;br/&gt; autoclavable&lt;br/&gt; fiber optic light transmission incorporated</td><td>Karl Storz</td><td>64301AA</td><td></td></tr><tr><td>Protection and Examination Sheath</td><td>Karl Storz</td><td>61029C</td><td></td></tr></tbody></table>

a genetically engineered mouse model of sporadic colorectal cancer

尽管具有易于适用性和成本效益的优点，但是基于肿瘤细胞注射的结直肠癌小鼠模型具有严重的局限性，并且不能准确模拟肿瘤生物学和肿瘤细胞传播。引入基因工程小鼠模型来克服这些局限性;然而，这种模型在技术上是苛刻的，特别是在大器官如结肠中仅需要单个肿瘤。 结果，开发了免疫活性的基因工程的结肠直肠癌小鼠模型，其发展高度均匀的肿瘤，并且可以用于肿瘤生物学研究以及治疗试验。肿瘤发展是通过复合条件突变小鼠中腺瘤病毒的远端结肠的手术，节段性感染开始的。可以通过结肠镜检查容易地检测和监测肿瘤。我们这里描述了节段性腺病毒感染的外科技术结肠， 通过高分辨率结肠镜检查监测肿瘤，并呈现所得的结肠直肠肿瘤。

如果充分进行，&gt; 85％的动物会发展成肿瘤。这里介绍的外科手术的死亡率&lt;5％，结肠镜检查的死亡率几乎不存在。在大多数小鼠中，检测到单个病变;在大约30％的2〜3个小腺瘤中，可以检测到肿瘤诱导后2-3周内通常融合到单个肿瘤。 所得肿瘤的表型和生物学行为高度依赖于动物的条件突变。 Apc的局部cre介导的敲除足以诱?...

Watch this Scientific Journal Video about A Genetically Engineered Mouse Model of Sporadic Colorectal Cancer at JoVE.com

A Genetically Engineered Mouse Model of Sporadic Colorectal Cancer

遗传工程小鼠模型的散发性结肠直肠癌

提出了通过节段性腺病毒感染建立结肠直肠癌基因工程小鼠模型的方法及其通过高分辨率结肠镜检查进行的监测。

Despite the advantages of easy applicability and cost-effectiveness, colorectal cancer mouse models based on tumor cell injection have severe limitations ...

a-genetically-engineered-mouse-model-of-sporadic-colorectal-cancer

Universidad de Cartagena UDEC

Research

JoVE Journal

Cancer Research

9.4K 次观看.  Technische Universität Dresden. 提出了通过节段性腺病毒感染建立结肠直肠癌基因工程小鼠模型的方法及其通过高分辨率结肠镜检查进行的监测。 

视频: A Genetically Engineered Mouse Model of Sporadic Colorectal Cancer

Isolation of Circulating Tumor Cells in an Orthotopic Mouse Model of Colorectal Cancer

Despite the advantages of easy applicability and cost-effectiveness, subcutaneous mouse models have severe limitations and do not accurately simulate tumor biology and tumor cell dissemination. Orthotopic mouse models have been introduced to overcome these limitations; however, such models are technically demanding, especially in hollow organs such as the large bowel. In order to produce uniform tumors which reliably grow and metastasize, standardized techniques of tumor cell preparation and injection are critical. 
We have developed an orthotopic mouse model of colorectal cancer (CRC) which develops highly uniform tumors and can be used for tumor biology studies as well as therapeutic trials. Tumor cells from either primary tumors, 2-dimensional (2D) cell lines or 3-dimensional (3D) organoids are injected into the cecum and, depending on the metastatic potential of the injected tumor cells, form highly metastatic tumors. In addition, CTCs can be found regularly. We here describe the technique of tumor cell preparation from both 2D cell lines and 3D organoids as well as primary tumor tissue, the surgical and injection techniques as well as the isolation of CTCs from the tumor-bearing mice, and present tips for troubleshooting.

We describe the establishment of orthotopic colorectal tumors via injection of tumor cells or organoids into the cecum of mice and the subsequent isolation of circulating tumor cells (CTCs) from this model.

在直肠癌原位小鼠模型中循环肿瘤细胞的分离

Despite the advantages of easy applicability and cost-effectiveness, subcutaneous mouse models have severe limitations and do not accurately simulate ...

科研

癌症研究

Orthotopic Mouse Model of Colorectal Cancer

The traditional subcutaneous tumor model is less than ideal for studying colorectal cancer. Orthotopic mouse models of colorectal cancer, which feature cancer cells growing in their natural location, replicate human disease with high fidelity. Two techniques can be used to establish this model. Both techniques are similar and require mouse anesthesia and laparotomy for exposure of the cecum. One technique involves injection of a colorectal cancer cell suspension into the cecal wall. Cancer cells are first grown in culture, harvested when subconfluent and prepared as a single cell suspension. A small volume of cells is injected slowly to avoid leakage. The other technique involves transplantation of a piece of subcutaneous tumor onto the cecum. A mouse with a previously established subcutaneous colorectal tumor is euthanized and the tumor is removed using sterile technique. The tumor piece is divided into small pieces for transplantation to another mouse. Prior to transplantation, the cecal wall is lightly damaged to facilitate tumor cell infiltration. The time to developing primary tumors and liver metastases will vary depending on the technique, cell line, and mouse species used. This orthotopic mouse model is useful for studying the natural progression of colorectal cancer and testing new therapeutic agents against colorectal cancer.

Two techniques can be used to establish this model: injection of a cancer cell suspension into the cecal wall or transplantation of a piece of subcutaneous tumor onto the cecum. This model is useful for studying the natural progression of colorectal cancer and testing new therapeutic agents against colorectal cancer.

结直肠癌原位小鼠模型

The traditional subcutaneous tumor model is less than ideal for studying colorectal cancer. Orthotopic mouse models of colorectal cancer, which feature ...

生物学

Modeling Colitis-Associated Cancer with Azoxymethane (AOM) and Dextran Sulfate Sodium (DSS)

Individuals with inflammatory bowel disease (IBD), such as Crohn's disease (CD) or ulcerative colitis (UC) are at increased risk of developing colorectal cancer (CRC) over healthy individuals. This risk is proportional to the duration and extent of disease, with a cumulative incidence as high as 30% in individuals with longstanding UC with widespread colonic involvement.1 Colonic dysplasia in IBD and colitis associated cancer (CAC) are believed to develop as a result of repeated cycles of epithelial cell injury and repair while these cells are bathed in a chronic inflammatory cytokine milieu.2 While spontaneous and colitis-associated cancers share the quality of being adenocarcinomas, the sequence of underlying molecular events is believed to be different.3 This distinction argues the need for specific animal models of CAC.
Several mouse models currently exist for the study of CAC. Dextran sulfate sodium (DSS), an agent with direct toxic effects on the colonic epithelium, can be administered in drinking water to mice in multiple cycles to create a chronic inflammatory state. With sufficient duration, some of these mice will develop tumors.4 Tumor development is hastened in this model if administered in a pro-carcinogenic setting. These include mice with genetic mutations in tumorigenesis pathways (APC, p53, Msh2), as well as mice pre-treated with genotoxic agents (azoxymethane [AOM], 1,2-dimethylhydrazine [DMH]).5 
The combination of DSS with AOM as a model for colitis associated cancer has gained popularity for its reproducibility, potency, low price, and ease of use. Though they have a shared mechanism, AOM has been found to be more potent and stable in solution than DMH. While tumor development in other models generally requires several months, mice injected with AOM and subsequently treated with DSS develop adequate tumors in as little as 7-10 weeks.6, 7 Finally, AOM and DSS can be administered to mice of any genetic background (knock out, transgenic, etc.) without cross-breeding to a specific tumorigenic strain. Here, we demonstrate a protocol for inflammation-driven colonic tumorigenesis in mice utilizing a single injection of AOM followed by three seven-day cycles of DSS over a 10 week period. This model induces tumors with histological and molecular changes closely resembling those occurring in human CAC and provides a highly valuable model for the study of oncogenesis and chemoprevention in this disease.8

Modeling Colitis-Associated Cancer with Azoxymethane AOM and Dextran Sulfate Sodium DSS

We demonstrate a protocol in which administration of the genotoxic agent azoxymethane (AOM) followed by three cycles of the pro-inflammatory agent dextran sulfate sodium (DSS) rapidly and consistently generates colon tumors in mice with morphologic and molecular similarities to those seen in human colitis-associated cancer.

建模结肠炎相关的癌症与​​氧化偶氮甲烷（AOM）和葡聚糖硫酸钠（DSS）

Individuals with inflammatory bowel disease (IBD), such as Crohn's disease (CD) or ulcerative colitis (UC) are at increased risk of developing colorectal ...

医学

Generation of Tumor Organoids from Genetically Engineered Mouse Models of Prostate Cancer

Methods based on homologous recombination to modify genes have significantly furthered biological research. Genetically engineered mouse models (GEMMs) are a rigorous method for studying mammalian development and disease. Our laboratory has developed several GEMMs of prostate cancer (PCa) that lack expression of one or multiple tumor suppressor genes using the site-specific Cre-loxP recombinase system and a prostate-specific promoter. In this article, we describe our method for necropsy of these PCa GEMMs, primarily focusing on dissection of mouse prostate tumors. New methods developed over the last decade have facilitated the culture of epithelial-derived cells to model organ systems in vitro in three dimensions. We also detail a 3D cell culture method to generate tumor organoids from mouse PCa GEMMs. Pre-clinical cancer research has been dominated by 2D cell culture and cell line-derived or patient-derived xenograft models. These methods lack tumor microenvironment, a limitation of using these techniques in pre-clinical studies. GEMMs are more physiologically-relevant for understanding tumorigenesis and cancer progression. Tumor organoid culture is an in vitro model system that recapitulates tumor architecture and cell lineage characteristics. In addition, 3D cell culture methods allow for growth of normal cells for comparison to tumor cell cultures, rarely possible using 2D cell culture techniques. In combination, use of GEMMs and 3D cell culture in pre-clinical studies has the potential to improve our understanding of cancer biology.&#160;

We show a method for necropsy and dissection of mouse prostate cancer models, focusing on prostate tumor dissection. A step-by-step protocol for generation of mouse prostate tumor organoids is also presented.

从前列腺癌的遗传工程小鼠模型生成肿瘤有机体

Methods based on homologous recombination to modify genes have significantly furthered biological research. Genetically engineered mouse models (GEMMs) ...

遗传学

Molecular and Immunologic Techniques in a Genetically Engineered Mouse Model of Gastrointestinal Stromal Tumor

Gastrointestinal stromal tumor (GIST) is the most common human sarcoma and is typically driven by a single mutation in the KIT receptor. Across tumor types, numerous mouse models have been developed in order to investigate the next generation of cancer therapies. However, in GIST, most in vivo studies use xenograft mouse models which have inherent limitations. Here, we describe an immunocompetent, genetically engineered mouse model of gastrointestinal stromal tumor harboring a KitV558&#916;/+ mutation. In this model, mutant KIT, the oncogene responsible for most GISTs, is driven by its endogenous promoter leading to a GIST which mimics the histological appearance and immune infiltrate seen in human GISTs. Furthermore, this model has been used successfully to investigate both targeted molecular and immune therapies. Here, we describe the breeding and maintenance of a KitV558&#916;/+ mouse colony. Additionally, this paper details the treatment and procurement of GIST, draining mesenteric lymph node, and adjacent cecum in KitV558&#916;/+ mice, as well as sample preparation for molecular and immunologic analyses.

The goal of this manuscript is to describe the KitV558&#916;/+&#160;mouse model and techniques for successful dissection and processing of mouse specimens.

分子和免疫技术在胃肠道间质瘤基因工程小鼠模型中

Gastrointestinal stromal tumor (GIST) is the most common human sarcoma and is typically driven by a single mutation in the KIT receptor. Across tumor ...

Initiation of Metastatic Breast Carcinoma by Targeting of the Ductal Epithelium with Adenovirus-Cre: A Novel Transgenic Mouse Model of Breast Cancer

Breast cancer is a heterogeneous disease involving complex cellular interactions between the developing tumor and immune system, eventually resulting in exponential tumor growth and metastasis to distal tissues and the collapse of anti-tumor immunity. Many useful animal models exist to study breast cancer, but none completely recapitulate the disease progression that occurs in humans. In order to gain a better understanding of the cellular interactions that result in the formation of latent metastasis and decreased survival, we have generated an inducible transgenic mouse model of YFP-expressing ductal carcinoma that develops after sexual maturity in immune-competent mice and is driven by consistent, endocrine-independent oncogene expression. Activation of YFP, ablation of p53, and expression of an oncogenic form of K-ras was achieved by the delivery of an adenovirus expressing Cre-recombinase into the mammary duct of sexually mature, virgin female mice. Tumors begin to appear 6 weeks after the initiation of oncogenic events. After tumors become apparent, they progress slowly for approximately two weeks before they begin to grow exponentially. After 7-8 weeks post-adenovirus injection, vasculature is observed connecting the tumor mass to distal lymph nodes, with eventual lymphovascular invasion of YFP+ tumor cells to the distal axillary lymph nodes. Infiltrating leukocyte populations are similar to those found in human breast carcinomas, including the presence of &#945;&#946; and &#947;&#948; T cells, macrophages and MDSCs. This unique model will facilitate the study of cellular and immunological mechanisms involved in latent metastasis and dormancy in addition to being useful for designing novel immunotherapeutic interventions to treat invasive breast cancer.

Activation of latent mutations with adenovirus-Cre into the mammary ductal system results in a clinically relevant metastatic breast cancer. Incorporation of a YFP promoter allows tracking of distal metastatic tumor cells. This model is useful to study latent metastasis, anti-tumor immunity, and for designing novel immunotherapies to treat breast cancer.

乳腺癌的一种新的转基因小鼠模型：转移性乳腺癌通过瞄准导管上皮与腺病毒Cre重组酶的启动

Breast cancer is a heterogeneous disease involving complex cellular interactions between the developing tumor and immune system, eventually resulting in ...

Modeling Prostate Cancer in Genetically-engineered Mouse Models: A CRISPR/Cas9-mediated Localized Gene Editing Technique in Mouse Anterior Prostate Lobe Cells

Source: Riedel, M. et al. <a href="https://jove.unicartagenaproxy.elogim.com/video/57525" target="_blank">Virus Delivery of CRISPR Guides to the Murine Prostate for Gene Alteration.</a> J. Vis. Exp. (2018)
This video describes a method for editing specific genes in prostate gland cells using an adenovirus-based delivery system. The approach allows for orthotopic and localized alteration of gene expression utilizing CRISPR technology to develop novel mouse models for prostate cancer.

基因工程小鼠模型中的前列腺癌建模:小鼠前前列腺叶细胞中的 CRISPR/Cas9 介导的局部基因编辑技术

Source: Riedel, M. et al. Virus Delivery of CRISPR Guides to the Murine Prostate for Gene Alteration. J. Vis. Exp. (2018)
This video describes a method ...

JoVE Encyclopedia of Experiments

Prostate Cancer

Animal model studies

Segmental Adeno-Cre Infection: A Technique to Generate Isolated Colorectal Cancer using Genetically Engineered Mouse Models

Source: Betzler, A. M., et al. <a href="https://jove.unicartagenaproxy.elogim.com/v/55952"> A Genetically Engineered Mouse Model of Sporadic Colorectal Cancer.</a>&#160;J. Vis. Exp. (2017).
This video describes the generation of a colorectal mouse model by segmental adeno-cre infection. The infected colon segment leads to tumorigenesis within this segment of the mucosa, resulting in adenomas ultimately progressing to invasive and metastatic carcinoma. This model is an attractive platform for studies of cancer biology and preclinical therapeutic trials.

节段性腺-Cre 感染:一种使用基因工程小鼠模型生成孤立性结直肠癌的技术

Source: Betzler, A. M., et al.  A Genetically Engineered Mouse Model of Sporadic Colorectal Cancer.&#160;J. Vis. Exp. (2017).
This video describes the ...

Colorectal Cancer

Spontaneous Metastasis Mouse Models: A Platform to Study the Metastatic Potential of Orthotopically Injected Colorectal Cancer Cells

Source: Okazawa, Y., et al. <a href="https://jove.unicartagenaproxy.elogim.com/v/57374"> High-sensitivity Detection of Micrometastases Generated by GFP Lentivirus-transduced Organoids Cultured from a Patient-derived Colon Tumor.</a>&#160;J. Vis. Exp.&#160;(2018).
This video describes the protocol to establish a spontaneous metastasis mouse model of colorectal cancer patient-derived xenografts or PDXs. These xenograft models partially mimic the features of human carcinomas and understand the critical aspects of human malignancies, including invasion and metastasis.&#160;

自发性转移小鼠模型:研究原位注射的结直肠癌细胞转移潜力的平台

Source: Okazawa, Y., et al.  High-sensitivity Detection of Micrometastases Generated by GFP Lentivirus-transduced Organoids Cultured from a ...

Generating PDOX Mouse Model of Colorectal Cancer: Studying Progression of Colorectal Cancer in a Murine Model

Source: &#160;Moret, R. et al. <a href="https://jove.unicartagenaproxy.elogim.com/v/59223"> Patient-derived Orthotopic Xenograft Models for Human Urothelial Cell Carcinoma and Colorectal Cancer Tumor Growth and Spontaneous Metastasis.</a> J. Vis. Exp. (2019).
This video describes the protocol for generating a patient-derived orthotopic xenograft colorectal cancer mouse model to study the progression of colorectal cancer cells in non-obese diabetic/severe combined immunodeficiency (NOD/SCID) mice. This murine model helps in understanding primary tumor growth and metastases pattern.

生成结直肠癌的 PDOX 小鼠模型:在小鼠模型中研究结直肠癌的进展

Source: &#160;Moret, R. et al.  Patient-derived Orthotopic Xenograft Models for Human Urothelial Cell Carcinoma and Colorectal Cancer Tumor Growth and ...

遗传工程小鼠模型的散发性结肠直肠癌

摘要

探索更多视频

此视频中的章节

在直肠癌原位小鼠模型中循环肿瘤细胞的分离

结直肠癌原位小鼠模型

建模结肠炎相关的癌症与氧化偶氮甲烷（AOM）和葡聚糖硫酸钠（DSS）

从前列腺癌的遗传工程小鼠模型生成肿瘤有机体

分子和免疫技术在胃肠道间质瘤基因工程小鼠模型中

乳腺癌的一种新的转基因小鼠模型：转移性乳腺癌通过瞄准导管上皮与腺病毒Cre重组酶的启动

基因工程小鼠模型中的前列腺癌建模:小鼠前前列腺叶细胞中的 CRISPR/Cas9 介导的局部基因编辑技术

节段性腺-Cre 感染:一种使用基因工程小鼠模型生成孤立性结直肠癌的技术

自发性转移小鼠模型:研究原位注射的结直肠癌细胞转移潜力的平台

生成结直肠癌的 PDOX 小鼠模型:在小鼠模型中研究结直肠癌的进展

遗传工程小鼠模型的散发性结肠直肠癌

摘要

探索更多视频

此视频中的章节

在直肠癌原位小鼠模型中循环肿瘤细胞的分离

结直肠癌原位小鼠模型

建模结肠炎相关的癌症与​​氧化偶氮甲烷（AOM）和葡聚糖硫酸钠（DSS）

从前列腺癌的遗传工程小鼠模型生成肿瘤有机体

分子和免疫技术在胃肠道间质瘤基因工程小鼠模型中

乳腺癌的一种新的转基因小鼠模型：转移性乳腺癌通过瞄准导管上皮与腺病毒Cre重组酶的启动

基因工程小鼠模型中的前列腺癌建模:小鼠前前列腺叶细胞中的 CRISPR/Cas9 介导的局部基因编辑技术

节段性腺-Cre 感染:一种使用基因工程小鼠模型生成孤立性结直肠癌的技术

自发性转移小鼠模型:研究原位注射的结直肠癌细胞转移潜力的平台

生成结直肠癌的 PDOX 小鼠模型:在小鼠模型中研究结直肠癌的进展

建模结肠炎相关的癌症与氧化偶氮甲烷（AOM）和葡聚糖硫酸钠（DSS）