Name: tumor allotransplantation in drosophila melanogaster with a programmable auto-nanoliter injector
Uploaded: 2021-02-02T14:30:00
Description: Watch this Scientific Journal Video about Tumor Allotransplantation in Drosophila melanogaster with a Programmable Auto-Nanoliter Injector at JoVE.com

我们感谢前实验室成员杨胜安博士和胡安-马丁·波蒂拉先生在制定该协议方面所做的贡献。我们非常感谢Yan Song博士在北京大学生命科学学院的实验室分享他们的手动异体移植方案。我们还感谢考尔德·埃尔斯沃思先生和珠穆朗玛峰·夏皮罗先生对手稿的批判性阅读。
大规模杀伤性武器获得了美国国立卫生研究院（https://www.nih.gov/）的资助（GM072562、CA224381、CA227789）和美国国家科学基金会（htps://nsf.gov/）的资助（IOS-155790）。资助者在研究设计、数据收集和分析、发表决定或手稿准备方面没有任何作用。

1. SG假想环肿瘤的制备
<ol><li>具有 UAS-NICD 基因型（雄性：10-15只苍蝇）和 阿克特-加仑4，UAS-GFP / CyO;浴缸 -Gal80ts （处女雌性：10-15只苍蝇）的杂交成蝇，并允许它们在18°C下繁殖1天。 选定的成年苍蝇应为5-9天大，以确保高生育能力。</li>
	<li>让成蝇在18°C下在小瓶中含有的苍蝇食物中产卵24小时，然后除去成蝇。 注意：苍蝇食物是使用 果蝇 库存中心8的标准玉米粉食...

<ol>
	<li>Rossi, F., Gonzalez, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Studying+tumor+growth+in+Drosophila+using+the+tissue+allograft+method.">Studying tumor growth in Drosophila using the tissue allograft method.</a> Nature Protocols. 10 (10), 1525-1534 (2015).</li><li>Magadi, S. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dissecting+Hes-centred+transcriptional+networks+in+neural+stem+cell+maintenance+and+tumorigenesis+in+Drosophilia.">Dissecting Hes-centred transcriptional networks in neural stem cell maintenance and tumorigenesis in Drosophilia.</a> Development. 147 (22), (2020).</li><li>Haller, S., Limmer, S., Ferrandon, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Pseudomonas Methods and Protocols. , 723-740 (2014).</li><li>Letini&#263;, B., Kemp, A., Christian, R., Koekemoer, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inoculation+protocol+for+the+African+malaria+vector,+Anopheles+arabiensis,+by+means+of+nano-injection.">Inoculation protocol for the African malaria vector, Anopheles arabiensis, by means of nano-injection.</a> African Entomology. 26 (2), 422-428 (2018).</li><li>Mejia, M., Heghinian, M. D., Busch, A., Mar&#237;, F., Godenschwege, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Paired+nanoinjection+and+electrophysiology+assay+to+screen+for+bioactivity+of+compounds+using+the+Drosophila+melanogaster+giant+fiber+system.">Paired nanoinjection and electrophysiology assay to screen for bioactivity of compounds using the Drosophila melanogaster giant fiber system.</a> Journal of Visualized Experiments: JoVE. (62), e3597 (2012).</li><li>Miles, W. O., Dyson, N. J., Walker, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modeling+tumor+invasion+and+metastasis+in+Drosophila.">Modeling tumor invasion and metastasis in Drosophila.</a> Disease Models &amp; Mechanisms. 4 (6), 753 (2011).</li><li>Yang, S. A., Portilla, J. M., Mihailovic, S., Huang, Y. C., Deng, W. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oncogenic+notch+triggers+neoplastic+tumorigenesis+in+a+transition-zone-like+tissue+microenvironment.">Oncogenic notch triggers neoplastic tumorigenesis in a transition-zone-like tissue microenvironment.</a> Developmental Cell. 49 (3), 461-472 (2019).</li><li>Bloomington Drosophila Stock Center. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> BDSC Cornmeal Food. , (2020).</li><li>Garelli, A., Gontijo, A. M., Miguela, V., Caparros, E., Dominguez, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaginal+discs+secrete+insulin-like+peptide+8+to+mediate+plasticity+of+growth+and+maturation.">Imaginal discs secrete insulin-like peptide 8 to mediate plasticity of growth and maturation.</a> Science. 336 (6081), 579-582 (2012).</li><li>Kennison, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dissection+of+larval+salivary+glands+and+polytene+chromosome+preparation.">Dissection of larval salivary glands and polytene chromosome preparation.</a> CSH Protocols. 2008, (2008).</li><li>Ji, H., Han, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=LarvaSPA,+a+method+for+mounting+drosophila+larva+for+long-term+time-lapse+imaging.">LarvaSPA, a method for mounting drosophila larva for long-term time-lapse imaging.</a> Journal of Visualized Experiments: JoVE. (156), (2020).</li><li>Mirzoyan, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drosophila+melanogaster:+a+model+organism+to+study+cancer.">Drosophila melanogaster: a model organism to study cancer.</a> Frontiers in Genetics. 10, 51 (2019).</li><li>Bangi, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drosophila+at+the+intersection+of+infection,+inflammation,+and+cancer.">Drosophila at the intersection of infection, inflammation, and cancer.</a> Frontiers in Cellular and Infection Microbiology. 3, 103 (2013).</li><li>Saavedra, P., Perrimon, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drosophila+as+a+model+for+tumor-induced+organ+wasting.">Drosophila as a model for tumor-induced organ wasting.</a> Advances in Experimental Medicine and Biology. 1167, 191-205 (2019).</li><li>Figueroa-Clarevega, A., Bilder, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Malignant+drosophila+tumors+interrupt+insulin+signaling+to+induce+cachexia-like+wasting.">Malignant drosophila tumors interrupt insulin signaling to induce cachexia-like wasting.</a> Developmental Cell. 33 (1), 47-55 (2015).</li><li>Koyama, L. A. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bellymount+enables+longitudinal,+intravital+imaging+of+abdominal+organs+and+the+gut+microbiota+in+adult+Drosophila.">Bellymount enables longitudinal, intravital imaging of abdominal organs and the gut microbiota in adult Drosophila.</a> PLOS Biology. 18 (1), 3000567 (2020).</li></ol>

肿瘤同种异体移植可以帮助研究人员解决果蝇肿瘤生长和进展过程中出现的某些问题。其中一个挑战是规避在原发性肿瘤培养期间携带肿瘤的幼虫或成虫的过早死亡12。在这种情况下，持续的肿瘤同种异体移植允许肿瘤无限生长，这有助于肿瘤生长，转移和进化的纵向研究。肿瘤同种异体移植也可用于评估宿主肿瘤相互作用的各个方面7，<sup class=...

该方案为果 蝇 幼虫唾液腺（SG）假想环肿瘤的同种异体移植到使用自动纳升注射装置（例如，Nanoject）的成年宿主的腹部提供了分步指导。该方案还为随后将肿瘤重新同种异体移植到新一代成年宿主中提供了方向，这为继续纵向研究肿瘤特征提供了机会，例如肿瘤进化和肿瘤 - 宿主相互作用。该协议也可以应用于药物筛选实验。
该方法的开发是为了提高使用手动注射器1在果蝇中进行肿瘤同种异体移植的疗效，其吸力和注射力通常不一致，导致肿瘤同种异体移植的次优结果。自动注射器装置提供更好的控制，并可降低同种异体移植物后的苍蝇死亡率。训练有素的操作员使用自动注射器可以实现超过90%的宿主存活率，而使用手动注射器时约为80%1。同种异体移植后第8-12天的总肿瘤获得率为60%-80%。平均注射时间也从使用手动注射器的每只苍蝇30-40秒提高到使用自动注射器的每只苍蝇20-25秒。
该协议是在 果蝇 肿瘤同种异体移植中使用自动注射器装置的首批几个方案之一。最近的一项研究还使用自体注射器进行肿瘤神经干细胞的同种异体移植2。以前，在 果蝇 中使用自动注射器来研究细菌毒力3，寄生虫感染和宿主防御4，以及筛查不同化合物的生物活性5。我们的方案使自动注射器装置适应肿瘤注射使用，并寻求为 果蝇 研究人员提供更高质量和更一致的结果，同时节省他们相当大的时间。该方案不仅可以用于肿瘤的同种异体移植，还可以针对类似口径的野生型和突变组织的同种异体移植进行定制6。
该方案中使用的果蝇NICD肿瘤最初是由Yang等人引入的，SG假想环过渡区是一个“肿瘤热点”，表现出高水平的内源性Janus激酶/信号传感器和转录激活剂（JAK-STAT）和c-Jun N-末端激酶（JNK）活性。此外，过渡区具有高水平的基质金属蛋白酶-1（MMP1）7，这使得该区域特别有利于肿瘤发生。仅通过NICD过表达激活缺口通路就足以持续启动肿瘤形成。这些肿瘤随后可以进行同种异体移植，以允许研究广泛的主题，包括肿瘤细胞分裂，侵袭和肿瘤 - 宿主相互作用。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Confocal Laser Scanning Microscope</td>
			<td>Zeiss</td>
			<td>LSM 980</td>
			<td>Also known as &#34;Zeiss LSM 980&#34;</td>
		</tr>
		<tr>
			<td>Cornmeal Fly Food</td>
			<td>Bloomington Drosophila Stock Center</td>
			<td>N/A</td>
			<td>Also known as &#34;BDSC Standard Cornmeal Food&#34;</td>
		</tr>
		<tr>
			<td>Dissection Needle (30Gx1/2)</td>
			<td>BD PrecisionGlide</td>
			<td>305106</td>
			<td></td>
		</tr>
		<tr>
			<td>Dissection Plate</td>
			<td>Fisher Scientific</td>
			<td>12-565B</td>
			<td></td>
		</tr>
		<tr>
			<td>Fly Tape</td>
			<td>Fisherbrand</td>
			<td>159015A</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluoresence Adapter for Stero Microscope</td>
			<td>Electron Microscopy Sciences</td>
			<td>SFA-UV</td>
			<td>Also known as &#34;NightSea Fluorescence Adapter&#34;</td>
		</tr>
		<tr>
			<td>Fluoresence Microscope</td>
			<td>Zeiss</td>
			<td>495015-0001-000</td>
			<td>Also known as &#34;Zeiss Stereo Discovery.V8&#34;</td>
		</tr>
		<tr>
			<td>Forceps</td>
			<td>Fine Science Tools</td>
			<td>11251-10</td>
			<td>Also known as &#34;Dumont #5 Forceps&#34;&#160;</td>
		</tr>
		<tr>
			<td>Glass Capillary (3.5&#39;&#39;)</td>
			<td>Drummond</td>
			<td>3-000-203-G/X</td>
			<td></td>
		</tr>
		<tr>
			<td>Glue</td>
			<td>Elmer</td>
			<td>E305</td>
			<td>Also known as &#34;Elmer Washabale Clear Glue&#34;</td>
		</tr>
		<tr>
			<td>Light Microscope</td>
			<td>Zeiss</td>
			<td>435063-9010-100</td>
			<td>Also known as &#34;Zeiss Stemi 305&#34;</td>
		</tr>
		<tr>
			<td>Micropipette Puller</td>
			<td>World Precision Instruments</td>
			<td>PUL-1000</td>
			<td>Also known as &#34;Four Step Micropipette Puller&#34;</td>
		</tr>
		<tr>
			<td>Nanoject Apparatus</td>
			<td>Drummond</td>
			<td>3-000-204</td>
			<td>Also known as &#34;Nanoject II Auto-Nanoliter Injector&#34;</td>
		</tr>
		<tr>
			<td>Schneider&#39;s Medium</td>
			<td>ThermoFisher</td>
			<td>21720001</td>
			<td></td>
		</tr>
		<tr>
			<td>Syringe (27G x1/2)</td>
			<td>BD PrecisionGlide</td>
			<td>305109</td>
			<td></td>
		</tr>
		<tr>
			<td>Vial</td>
			<td>Fisherbrand</td>
			<td>AS507</td>
			<td></td>
		</tr>
	</tbody>
</table>

tumor allotransplantation in drosophila melanogaster with a programmable auto-nanoliter injector

该协议描述了使用自动纳升注射装置在 黑色腹果蝇 中对肿瘤的同种异体移植。 通过使用自动注射器设备，与使用手动注射器获得的移植结果相比，经过培训的操作员可以获得更有效和一致的移植结果。在这里，我们按时间顺序涵盖了主题：从 果蝇 线的交叉，到原发肿瘤的诱导和解剖，原发性肿瘤移植到新的成年宿主中，以及肿瘤的持续代际移植以进行扩展研究。作为演示，这里我们使用Notch细胞内结构域（NICD）过表达诱导的唾液腺假想环肿瘤进行世代移植。这些肿瘤可以首先在弱小唾液腺假想环内的过渡区微环境中可靠地诱导，然后在体内同种异体移植和培养，以研究持续的肿瘤生长，进化和转移。这种同种异体移植方法可用于潜在的药物筛选计划，以及研究肿瘤 - 宿主相互作用。

在这里，我们使用纳升注射自动注射装置对SG假想环肿瘤进行了世代同位异体移植，并用共聚焦激光扫描显微镜进行了随后的肿瘤活体成像，从而更深入地研究了肿瘤生长，肿瘤细胞迁移和肿瘤 - 宿主相互作用等主题。安装苍蝇时，将它们粘在显微镜载玻片上，并通过聚二甲基硅氧烷（PDMS）块11约束它们。
图5A 具有第 1代（G1）...

Watch this Scientific Journal Video about Tumor Allotransplantation in Drosophila melanogaster with a Programmable Auto-Nanoliter Injector at JoVE.com

Tumor Allotransplantation in Drosophila melanogaster with a Programmable Auto-Nanoliter Injector

该方案为 果蝇 肿瘤的初始和持续世代同种异体移植到成年宿主的腹部提供了详细的指导，用于研究肿瘤的各个方面。使用自动注射器设备，与传统的手动方法相比，研究人员可以提高效率和肿瘤产量。

使用可编程自动纳升注射器在 黑色果蝇 中植入肿瘤异体

This protocol describes the allotransplantation of tumors in Drosophila melanogaster using an auto-nanoliter injection apparatus. With the use of an ...

该协议是最早描述在果蝇肿瘤同种异体移植中使用自动注射器装置的协议之一。近年来越来越受欢迎的话题。该方法的主要优点是，它通过提高肿瘤同种异体移植的速度，一致性和产量，提高了果蝇癌症研究的效率。果蝇中的肿瘤同种异体移植是一种微妙的技术，可能很难掌握。要有耐心，相信实践是完美的。在杂交苍蝇并在18摄氏度下孵育卵六天后，将含有孵化幼虫的小瓶转移到设定在29摄氏度的孵化器中。根据性别对麻醉的野生型或突变的成年苍蝇进行分类。将一块五厘米长的苍蝇胶带固定在显微镜载玻片上，粘性面朝上。用镊子将苍蝇的翅膀粘在胶带上，使苍蝇固定下来。将控制器盒和自动注射器放在光学显微镜的相对侧。使用四步微型移液器拉拔器处理玻璃毛细管的一端，将其加热到狭窄的封闭端。使用镊子以大约60度角夹住毛细管，以制备3.5英寸的玻璃毛细管。轻轻拧下喷油器的盖子。按住空按钮以推进注射器针头，直到显示其总长度的70%至80%。使用注射器在玻璃毛细管中填充矿物油。选择其中一个幼虫并转移到装有100微升施耐德培养基的解剖板中，以准备SG假想环肿瘤解剖。使用一对镊子固定幼虫体的中段，用另一对镊子捏住幼虫头部并纵向施加拉伸力。定位并分离幼虫的Y形SG，然后通过去除相邻组织来解剖SG假想环肿瘤。通过按住填充按钮，用施耐德培养基填充玻璃毛细管，一直到顶部的0.5厘米段。找到原发性肿瘤并按下填充按钮，直到肿瘤被吸入毛细血管。确保肿瘤位于毛细血管的尖端或距离毛细血管尖端几毫米。找到一只固定在磁带上的成年苍蝇。然后用镊子轻轻按住下腹部，用毛细血管刺穿腹部的下侧角质层。按下空按钮，直到肿瘤进入新的宿主腹部。使用镊子，轻轻捏住主机的翅膀，将其从胶带上取下。然后将其放入装有新鲜食物的新小瓶中。在同种异体后约10至14天使用荧光显微镜筛查在宿主腹部生长的肿瘤，并使用两对镊子将生长的同种异体移植肿瘤从宿主中解剖出来。使用一对镊子压住腹部，另一对镊子切开腹部角质层，暴露同种异体移植的肿瘤。小心地将肿瘤从附着的宿主组织中分离出来。使用无菌针头将肿瘤解剖成适合毛细血管大小的小块。对新一代成年宿主重复此过程以完成肿瘤的同种异体移植。肿瘤发展的过程是通过第10天同种异体移植后在成人宿主腹部生长的第一代SG假想环肿瘤的实时成像来跟踪的。第六代SG假想环肿瘤在第10天占据了宿主腹部的大部分，同种异体移植后如图所示。该阶段的成像可能有助于揭示肿瘤生长模式及其迁移和侵袭行为。肿瘤移植是肿瘤进展、进化和转移纵向研究的一项重要技术。结果对于研究肿瘤宿主相互作用具有重要意义。该原型在使用药物Temodal的癌症药物筛选中具有巨大潜力。使用这种技术，研究人员将能够在调查中节省大量时间和精力。

Research

JoVE Journal

Cancer Research

Tumor Allotransplantation in Drosophila melanogaster with a Programmable Auto-Nanoliter Injector

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

Labeling of Breast Cancer Patient-derived Xenografts with Traceable Reporters for Tumor Growth and Metastasis Studies

可追踪记者乳腺癌患者来源的移植瘤贴标肿瘤生长和转移的研究

The use of preclinical models to study tumor biology and response to treatment is central to cancer research. Long-established human cell lines, and many ...

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 ...

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 ...

In Vivo EPR Assessment of pH, pO2, Redox Status, and Concentrations of Phosphate and Glutathione in the Tumor Microenvironment

In Vivo EPR Assessment of pH, pO2, Redox Status, and Concentrations of Phosphate and Glutathione in the Tumor Microenvironment

体内在肿瘤微环境中 pH 值、 pO2、氧化还原状态、磷酸盐和谷胱甘肽浓度的 EPR 评价

This protocol demonstrates the capability of low-field electron paramagnetic resonance (EPR)-based techniques in combination with functional paramagnetic ...

Digital Polymerase Chain Reaction Assay for the Genetic Variation in a Sporadic Familial Adenomatous Polyposis Patient Using the Chip-in-a-tube Format

数字聚合酶链反应法测定零星家族性腺瘤性息肉病患者的基因变异

The quantitative analysis of human genetic variation is crucial for understanding the molecular characteristics of serious medical conditions, such as ...

In Vivo Inhibition of MicroRNA to Decrease Tumor Growth in Mice

在微RNA的Vivo抑制下，降低小鼠的肿瘤生长

MicroRNAs (miRNAs) are important regulators of gene expression through their ability to destabilize mRNA and inhibit translation of target mRNAs. An ...

Predicting In Vivo Payloads Delivery using a Blood-brain Tumor-barrier in a Dish

利用血脑肿瘤屏障在盘中进行活体有效载荷传递预测

Highly selective by nature, the blood-brain barrier (BBB) is essential for the brain homeostasis in physiological conditions. However, in the context of ...

In Vitro Assay to Study Tumor-macrophage Interaction

体外测定研究肿瘤-巨噬菌体相互作用

Tumor associated macrophages (TAMs) account for a large percentage of cells in the tumor mass for different types of cancers. Glioblastoma (GBM), a ...

Analyzing Tumor Gene Expression Factors with the CorExplorer Web Portal

分析肿瘤基因表达因子与CorExplorer门户网站

Differential gene expression analysis is an important technique for understanding disease states. The machine learning algorithm CorEx has shown utility ...

使用可编程自动纳升注射器在 黑色果蝇 中植入肿瘤异体

使用可编程自动纳升注射器在黑色果蝇中植入肿瘤异体