分子和免疫技术在胃肠道间质瘤基因工程小鼠模型中
Summary
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。
Abstract
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。
Introduction
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。
Protocol
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 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…
Representative Results
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?…
Discussion
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?…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
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。
Materials
| 100 micron filter | EMSCO | 1194-2360 | |
| 1x RBC lysis buffer | Life Technologies | 00-4333-57 | |
| 3mL syringe | Thermo Fisher Scientific/BD Biosciences | 14823435 | |
| 4–15% Mini-PROTEAN TGX Precast Protein Gels, 10-well, 30 µl | Bio-Rad | 4561083 | |
| 4% Paraformaldehyde Solution | Thermo Fisher Scientific | AAJ19943K2 | |
| 40 micron filter | EMSCO | 1194-2340 | |
| 5M NaCl | Sigma Aldrich | S6546 | |
| 70 micron filter | EMSCO | 1194-2350 | |
| AKT antibody (C67E7) | Cell Signaling | 4691 | |
| C57BL/6J mice | The Jackson Laboratory | ||
| Collagenase IV | Sigma Aldrich | C5138 | |
| Complete mini edta free protease inhibitor | Thomas Scientific | C852A34 | |
| Countess II Automated Cell Counter | Thermo Fisher Scientific | ||
| Disposable Scalpels | Thermo Fisher Scientific/Exel International | 14-840-00 | |
| Dnase I | Thomas Scientific | C756V81 | |
| Dog1 antibody | abcam | ab64085 | |
| EDTA | Sigma Aldrich | E9884 | |
| ERK antibody (p44/42) | Cell Signaling | 9102 | |
| FBS | Thomas Scientific | C788U23 | |
| FIJI software | FIJI | https://imagej.net/software/fiji | |
| Fisherbrand 850 Homogenizer | Thermo Fisher Scientific | 15-340-169 | |
| HBSS | University of Pennsylvania Cell Center | ||
| Imatinib mesylate | Selleck Chemicals | S1026 | |
| KIT antibody (D13A2) | Cell Signaling | 3074 | |
| KitV558Δ/+ Genotyping | Transnetyx | ||
| Microcentrifuge tubes (1.5mL) | Thermo Fisher Scientific | 05-408-129 | |
| Mouse on Mouse Immunodetection Kit, Basic | Vector Laboratories | BMK-2202 | |
| Nitrocellulose Membrane, Precut, 0.45 µm | Rio-Rad | 1620145 | |
| Nonfat Dry Milk | Thermo Fisher Scientific | NC9121673 | |
| Nonidet P 40 Substitute | Sigma Aldrich | 74385 | |
| p-AKT antibody (S473) | Cell Signaling | 4060 | |
| p-ERK antibody (p44/42) | Cell Signaling | 9101 | |
| p-KIT antibody (Y719) | Cell Signaling | 3391 | |
| PMSF Protease Inhibitor | Thermo Fisher Scientific | 36978 | |
| Proeinase K | Thermo Fisher Scientific | BP170050 | |
| Round-Bottom Polystyrene Test (FACS) Tubes | Falcon/Thermo Fisher Scientific | 14-959-2A | |
| RPMI | University of Pennsylvania Cell Center | ||
| Sodium fluoride (NaF) | Sigma Aldrich | 201154 | |
| Sodium orthovanadate (Na3VO4) | Sigma Aldrich | S6508 | |
| SuperSignal West Dura Extended Duration Substrate | Thermo Fisher Scientific | 34076 | |
| TBS buffer (10x) | University of Pennsylvania Cell Center | ||
| Tissue culture dish (100mm2) | Thermo Fisher Scientific/Falcon | 08-772E | |
| TrisHCL | Thermo Fisher Scientific | BP1757500 | |
| Tween 20 | Rio-Rad | 1706531 | |
| vivaCT 80 platform | Scanco medical |
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Citazione di questo articolo
Tieniber, A. D., Hanna, A. N., Do, K., Wang, L., Rossi, F., DeMatteo, R. P. Molecular and Immunologic Techniques in a Genetically Engineered Mouse Model of Gastrointestinal Stromal Tumor. J. Vis. Exp. (183), e63853, doi:10.3791/63853 (2022).