Name: molecular and immunologic techniques in a genetically engineered mouse model of gastrointestinal stromal tumor
Uploaded: 2022-05-02T15:00:00
Description: Watch this Scientific Journal Video about Molecular and Immunologic Techniques in a Genetically Engineered Mouse Model of Gastrointestinal Stromal Tumor at JoVE.com

KitV558Δ/+ 小鼠经过基因工程改造，由彼得·贝斯梅尔博士10共享。这项工作得到了NIH拨款R01 CA102613和T32 CA251063的支持。

根据NIH指南和宾夕法尼亚大学IACUC的批准，所有小鼠都住在宾夕法尼亚大学的无病原体条件下。安乐死是按照宾夕法尼亚大学实验动物资源标准操作程序进行的。
1. 试剂盒V558Δ/+ 小鼠育种
<ol><li>使用 C57BL/6J 鼠标将套件 V558Δ/+ 鼠标反向交叉 10 次以上，放到 C57BL/6J 背景上。为此，将雄性 试剂盒V558Δ / +</sup...

<ol>
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试剂盒V558Δ/+小鼠模型是GIST分子和免疫学分析的强大研究工具。虽然育种策略需要单次杂交，但在分析肿瘤反应的实验中使用KitV558Δ/+小鼠队列需要广泛的育种。小鼠的年龄和性别应匹配，以确保相似的肿瘤重量，并且10%的小鼠在肿瘤建立时在8周龄之前死亡。如果使用先进的成像技术（如CT或MRI）来跟踪个体小鼠内的肿瘤体积，则可能采用不太广泛的育种策略。尽管?...

GIST是人类最常见的肉瘤，在美国发病率约为6，000例1。GIST似乎起源于称为Cajal间质细胞的胃肠道起搏器细胞，通常由酪氨酸激酶KIT或PDGFRA2中的单个突变驱动。手术是 GIST 的主要治疗方法，可以治愈，但晚期疾病患者可以使用酪氨酸激酶抑制剂 （TKI） 伊马替尼进行治疗。自20多年前推出以来，伊马替尼已经改变了GIST的治疗模式，将晚期疾病的生存率从1年提高到5年以上3，4，5。不幸的是，由于获得性KIT突变，伊马替尼很少能治愈，因此需要新的治疗方法来治疗这种肿瘤。
小鼠模型是研究癌症新疗法的重要研究工具。GIST6，7中已经开发并研究了多种皮下异种移植和患者来源的异种移植模型。然而，免疫缺陷小鼠不能完全代表人类GIST，因为GISTs根据其致癌突变具有不同的免疫谱，并且改变胃肠道肿瘤微环境可改善TKI治疗的效果8，9。KitV558Δ/+小鼠在 Kit 外显子 11 中具有杂合子种系缺失，该基因系对并列膜结构域进行编码，这是人类 GIST10 中最常见的突变位点。KitV558Δ/+小鼠发展出具有100%外显率的单个盲肠GIST，并且肿瘤具有与人类GIST8，11，12，13相似的组织学，分子信号传导，免疫浸润和对治疗的反应。在这里，我们描述了KitV558Δ / +小鼠中的育种，处理以及标本分离和处理，用于GIST中的分子和免疫学研究。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>100 micron filter</td>
			<td>EMSCO</td>
			<td>1194-2360</td>
			<td></td>
		</tr>
		<tr>
			<td>1x RBC lysis buffer</td>
			<td>Life Technologies</td>
			<td>00-4333-57</td>
			<td></td>
		</tr>
		<tr>
			<td>3mL syringe</td>
			<td>Thermo Fisher Scientific/BD Biosciences</td>
			<td>14823435</td>
			<td></td>
		</tr>
		<tr>
			<td>4&#8211;15% Mini-PROTEAN TGX Precast Protein Gels, 10-well, 30 &#181;l</td>
			<td>Bio-Rad</td>
			<td>4561083</td>
			<td></td>
		</tr>
		<tr>
			<td>4% Paraformaldehyde Solution</td>
			<td>Thermo Fisher Scientific</td>
			<td>AAJ19943K2</td>
			<td></td>
		</tr>
		<tr>
			<td>40 micron filter</td>
			<td>EMSCO</td>
			<td>1194-2340</td>
			<td></td>
		</tr>
		<tr>
			<td>5M NaCl</td>
			<td>Sigma Aldrich</td>
			<td>S6546</td>
			<td></td>
		</tr>
		<tr>
			<td>70 micron filter</td>
			<td>EMSCO</td>
			<td>1194-2350</td>
			<td></td>
		</tr>
		<tr>
			<td>AKT antibody (C67E7)</td>
			<td>Cell Signaling</td>
			<td>4691</td>
			<td></td>
		</tr>
		<tr>
			<td>C57BL/6J mice</td>
			<td>The Jackson Laboratory</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Collagenase IV</td>
			<td>Sigma Aldrich</td>
			<td>C5138</td>
			<td></td>
		</tr>
		<tr>
			<td>Complete mini edta free protease inhibitor</td>
			<td>Thomas Scientific</td>
			<td>C852A34</td>
			<td></td>
		</tr>
		<tr>
			<td>Countess II Automated Cell Counter</td>
			<td>Thermo Fisher Scientific</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Disposable Scalpels</td>
			<td>Thermo Fisher Scientific/Exel International</td>
			<td>14-840-00</td>
			<td></td>
		</tr>
		<tr>
			<td>Dnase I</td>
			<td>Thomas Scientific</td>
			<td>C756V81</td>
			<td></td>
		</tr>
		<tr>
			<td>Dog1 antibody</td>
			<td>abcam</td>
			<td>ab64085</td>
			<td></td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>Sigma Aldrich</td>
			<td>E9884</td>
			<td></td>
		</tr>
		<tr>
			<td>ERK antibody (p44/42)</td>
			<td>Cell Signaling</td>
			<td>9102</td>
			<td></td>
		</tr>
		<tr>
			<td>FBS</td>
			<td>Thomas Scientific</td>
			<td>C788U23</td>
			<td></td>
		</tr>
		<tr>
			<td>FIJI software</td>
			<td>FIJI</td>
			<td></td>
			<td>https://imagej.net/software/fiji</td>
		</tr>
		<tr>
			<td>Fisherbrand 850 Homogenizer</td>
			<td>Thermo Fisher Scientific</td>
			<td>15-340-169</td>
			<td></td>
		</tr>
		<tr>
			<td>HBSS</td>
			<td>University of Pennsylvania Cell Center</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Imatinib mesylate</td>
			<td>Selleck Chemicals</td>
			<td>S1026</td>
			<td></td>
		</tr>
		<tr>
			<td>KIT antibody (D13A2)</td>
			<td>Cell Signaling</td>
			<td>3074</td>
			<td></td>
		</tr>
		<tr>
			<td>KitV558&#916;/+ Genotyping</td>
			<td>Transnetyx</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Microcentrifuge tubes (1.5mL)</td>
			<td>Thermo Fisher Scientific</td>
			<td>05-408-129</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse on Mouse Immunodetection Kit, Basic</td>
			<td>Vector Laboratories</td>
			<td>BMK-2202</td>
			<td></td>
		</tr>
		<tr>
			<td>Nitrocellulose Membrane, Precut, 0.45 &#181;m</td>
			<td>Rio-Rad</td>
			<td>1620145</td>
			<td></td>
		</tr>
		<tr>
			<td>Nonfat Dry Milk</td>
			<td>Thermo Fisher Scientific</td>
			<td>NC9121673</td>
			<td></td>
		</tr>
		<tr>
			<td>Nonidet P 40 Substitute</td>
			<td>Sigma Aldrich</td>
			<td>74385</td>
			<td></td>
		</tr>
		<tr>
			<td>p-AKT antibody (S473)</td>
			<td>Cell Signaling</td>
			<td>4060</td>
			<td></td>
		</tr>
		<tr>
			<td>p-ERK antibody (p44/42)</td>
			<td>Cell Signaling</td>
			<td>9101</td>
			<td></td>
		</tr>
		<tr>
			<td>p-KIT antibody (Y719)</td>
			<td>Cell Signaling</td>
			<td>3391</td>
			<td></td>
		</tr>
		<tr>
			<td>PMSF Protease Inhibitor</td>
			<td>Thermo Fisher Scientific</td>
			<td>36978</td>
			<td></td>
		</tr>
		<tr>
			<td>Proeinase K</td>
			<td>Thermo Fisher Scientific</td>
			<td>BP170050</td>
			<td></td>
		</tr>
		<tr>
			<td>Round-Bottom Polystyrene Test (FACS) Tubes</td>
			<td>Falcon/Thermo Fisher Scientific</td>
			<td>14-959-2A</td>
			<td></td>
		</tr>
		<tr>
			<td>RPMI</td>
			<td>University of Pennsylvania Cell Center</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium fluoride (NaF)</td>
			<td>Sigma Aldrich</td>
			<td>201154</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium orthovanadate (Na3VO4)</td>
			<td>Sigma Aldrich</td>
			<td>S6508</td>
			<td></td>
		</tr>
		<tr>
			<td>SuperSignal West Dura Extended Duration Substrate</td>
			<td>Thermo Fisher Scientific</td>
			<td>34076</td>
			<td></td>
		</tr>
		<tr>
			<td>TBS buffer (10x)</td>
			<td>University of Pennsylvania Cell Center</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue culture dish (100mm2)</td>
			<td>Thermo Fisher Scientific/Falcon</td>
			<td>08-772E</td>
			<td></td>
		</tr>
		<tr>
			<td>TrisHCL</td>
			<td>Thermo Fisher Scientific</td>
			<td>BP1757500</td>
			<td></td>
		</tr>
		<tr>
			<td>Tween 20</td>
			<td>Rio-Rad</td>
			<td>1706531</td>
			<td></td>
		</tr>
		<tr>
			<td>&#160;vivaCT 80 platform</td>
			<td>Scanco medical</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

molecular and immunologic techniques in a genetically engineered mouse model of gastrointestinal stromal tumor

胃肠道间质瘤（GIST）是最常见的人肉瘤，通常由KIT受体中的单个突变驱动。在各种肿瘤类型中，已经开发了许多小鼠模型，以研究下一代癌症疗法。然而，在GIST中，大多数 体内 研究使用具有固有局限性的异种移植小鼠模型。在这里，我们描述了一种免疫功能正常的，基因工程化的胃肠道间质瘤小鼠模型，该模型含有 KitV558Δ / + 突变。在这个模型中，突变的KIT是负责大多数GIST的癌基因，由其内源性启动子驱动，导致GIST模仿人类GIST中的组织学外观和免疫浸润。此外，该模型已成功用于研究靶向分子和免疫疗法。在这里，我们描述了 KitV558Δ/+ 小鼠群落的繁殖和维护。此外，本文详细介绍了 KitV558Δ/+ 小鼠中GIST，引流肠系膜淋巴结和邻近盲肠的治疗和获取，以及用于分子和免疫学分析的样品制备。

KitV558Δ/+小鼠模型允许在免疫功能小鼠模型中研究治疗方法。由于进行性肠梗阻，KitV558Δ / +小鼠的平均寿命为8个月（图4）。来自KitV558Δ / +小鼠的肿瘤表达GIST的规范标志物，包括酪氨酸激酶KIT和跨膜通道DOG1（图5）以及转录因子ETV1（未显示）。可以研究肿?...

Watch this Scientific Journal Video about Molecular and Immunologic Techniques in a Genetically Engineered Mouse Model of Gastrointestinal Stromal Tumor at JoVE.com

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

本手稿的目的是描述 试剂盒V558Δ/+小鼠模型以及成功解剖和处理小鼠标本的技术。

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 ...

我们的方案描述了目前唯一使用的免疫活性小鼠模型GIST的育种，维护，解剖和标本处理。我们希望未来的研究人员能够利用这种小鼠模型，以便在GIST中进一步进行转化研究。GIST的传统疗法包括使用酪氨酸激酶抑制剂进行靶向分子治疗。该模型使研究人员能够在GIST中测试免疫疗法。首先，对所有器械进行消毒，在整个过程中戴上手套，并保持无菌状态。用70%乙醇准备切口区域。用剪刀做一个两厘米的中线垂直切口，进入腹腔。锐利地裂开任何腹腔内粘连。要切除引流肠系膜淋巴结，请识别盲肠并上位抬高其肠系膜。大约在结肠肠系膜底部的中间，识别肠系膜淋巴结并急剧解剖。根据需要，将淋巴结组织分成三分之一，用于蛋白质分离、组织学和单细胞悬浮液。将淋巴结组织置于20毫升RPMI培养基中以进行单细胞悬浮液并保持在冰上。盲肠在Kit558小鼠中大多被GIST取代。为了分离GIST和盲肠，小心地将回肠结肠连接处与肿瘤的底部分开。然后，再次将盲肠分开，在肿瘤基部远端两厘米处。在50%至60%的Kit558小鼠中，肿瘤的头部含有盲肠组织的盖子，其通常含有浆液，但可能很少含有脓液。尖锐地解剖帽状组织，使其远离肿瘤组织。将肿瘤组织和盲肠分成三部分，用于蛋白质分离、组织学和单细胞悬浮液。对于单细胞悬浮液，将肿瘤组织或盲肠置于含有2%FCS的HBSS中，足以覆盖样品，并保持在冰上。将RPMI培养基与淋巴结标本一起倒入100微米过滤器上。将过滤器移动到新的50毫升圆锥形，并用三毫升塑料注射器的软端捣碎淋巴结。用 20 毫升 RPMI 介质清洗过滤器。离心样品并吸出上清液。将沉淀重悬于20毫升珠缓冲液中，并倒在40微米过滤器上。收集细胞滤液。使用血细胞计数器计数细胞。离心样品并弃去上清液。然后，重悬于磁珠缓冲液中以进行流式细胞术。按照文本手稿中所述制备胶原酶缓冲液。将GIST放入无菌皿中，加入2.5毫升胶原酶缓冲液。使用无菌手术刀和剪刀，切碎肿瘤，直到它变成细小的碎片。使用大口径移液管将肿瘤和胶原酶吸入50毫升管中。以每分钟100转的速度在37摄氏度下孵育30分钟，然后用两毫升FBS淬灭反应。将胶原酶与GIST标本一起倒在100微米的过滤器上，并用三毫升塑料注射器的软端捣碎肿瘤并收集在50毫升管中。用20毫升HBSS清洗过滤器。将滤液在450 RCF下在四摄氏度下离心五分钟，然后弃去上清液。将沉淀重悬于20毫升珠缓冲液中，倒在40微米过滤器上收集滤液并使用血细胞计数器计数细胞。离心并吸出上清液，然后重悬于磁珠缓冲液中以进行流式细胞术。使用剪刀，纵向分开盲肠以暴露内粘膜。将其切成0.5厘米的部分，并将其放入50毫升的管中，其中含有5毫升的HBSS和2%的FBS。剧烈摇晃30秒，以450 RCF离心20秒。然后，丢弃上清液。加入 5 至 20 毫升 HBSS 和 2 毫摩尔 EDTA。在37摄氏度下以每分钟100转的速度孵育15分钟。离心并弃去上清液，然后将沉淀重悬于5至20毫升HBSS中。剧烈摇动混合物30秒，然后在450 RCF下离心20秒，弃去上清液。再次重复此过程。将沉淀重悬于5毫升胶原酶缓冲液中，然后在37摄氏度下以每分钟100转的速度孵育30分钟。每10分钟剧烈摇动一次，并用两毫升FBS淬灭反应。将胶原蛋白酶与盲肠标本倒在100微米的过滤器上，并用三毫升塑料注射器的软端捣碎。用20毫升HBSS洗涤过滤器并收集滤液。离心并弃去上清液。将沉淀重悬于20毫升珠缓冲液中，并倒在40微米过滤器上。收集滤液并使用血细胞计数器计数细胞。再次重复离心过程，并将沉淀重悬于珠缓冲液中以进行流式细胞术。由于进行性肠梗阻，Kit558小鼠的平均寿命为八个月。来自Kit558小鼠的肿瘤表达GIST的规范标志物，包括酪氨酸激酶KIT，跨膜通道Dog1和转录因子ETV1。研究了肿瘤中KIT信号通路的变化，例如下游标志物ERK和AKT或免疫微环境，其密切模仿人类GIST。MRI或CT用于跟踪肿瘤体积，作为肿瘤反应的准确测量。将肿瘤从回肠结肠连接处和盲肠帽剥离对于捕获真正的肿瘤重量以及免疫和分子分析非常重要。在GIST中，该模型允许研究肿瘤微环境以及免疫疗法。

Research

JoVE Journal

Cancer Research

The Drosophila Imaginal Disc Tumor Model: Visualization and Quantification of Gene Expression and Tumor Invasiveness Using Genetic Mosaics

The Drosophila Imaginal Disc Tumor Model: Visualization and Quantification of Gene Expression and Tumor Invasiveness Using Genetic Mosaics

该<em&gt;果蝇</em&gt;成虫盘瘤模型：基因的表达与肿瘤侵袭性的可视化和定量遗传马赛克

Drosophila melanogaster has emerged as a powerful experimental system for functional and mechanistic studies of tumor development and progression in the ...

科研

癌症研究

The Colon-26 Carcinoma Tumor-bearing Mouse as a Model for the Study of Cancer Cachexia

结肠-26癌荷瘤小鼠为癌症恶病质的研究模型

Cancer cachexia is the progressive loss of skeletal muscle mass and adipose tissue, negative nitrogen balance, anorexia, fatigue, inflammation, and ...

A Murine Orthotopic Bladder Tumor Model and Tumor Detection System

小鼠原位膀胱肿瘤模型和肿瘤检测系统

This protocol describes the generation of bladder tumors in female C57BL/6J mice using the murine bladder cancer cell line MB49, which has been modified ...

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

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 ...

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

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

Tumor Engraftment in a Xenograft Mouse Model of Human Mantle Cell Lymphoma

人地幔细胞淋巴瘤移植小鼠模型中肿瘤植入的研究

B lymphocytes are key players in immune cell circulation and they mainly home to and reside in lymphoid organs. While normal B cells only proliferate when ...

Utilizing High Resolution Ultrasound to Monitor Tumor Onset and Growth in Genetically Engineered Pancreatic Cancer Models

应用高分辨率超声监测基因工程胰腺癌的肿瘤发病和生长

The LSL-KrasG12D/+; LSL-Trp53R172H/+; Pdx-1-Cre (KPC) mouse model represents an established and frequently used transgenic model to evaluate novel ...

In Vitro Establishment of a Genetically Engineered Murine Head and Neck Cancer Cell Line using an Adeno-Associated Virus-Cas9 System

使用腺相关病毒-Cas9系统在Vitro建立基因工程的毛因头和颈部癌细胞系

The use of primary normal epithelial cells makes it possible to reproducibly induce genomic alterations required for cellular transformation by ...

A Novel Stromal Fibroblast-Modulated 3D Tumor Spheroid Model for Studying Tumor-Stroma Interaction and Drug Discovery

用于研究肿瘤-斯特罗马相互作用和药物发现的新型斯特罗姆纤维细胞-模组3D肿瘤球体模型

Tumor-stroma interactions play an important role in cancer progression. Three-dimensional (3D) tumor spheroid models are the most widely used in vitro ...

A Mouse Model to Investigate the Role of Cancer-Associated Fibroblasts in Tumor Growth

研究癌症相关成纤维细胞在肿瘤生长中的作用的小鼠模型

Cancer-associated fibroblasts (CAFs) can play an important role in tumor growth by creating a tumor-promoting microenvironment. Models to study the role ...