一个协议，用于基因诱导和良性和浸润性肿瘤可视化的头侧配合<em>果蝇</em
Summary
激活的致癌基因RAS一样的V12和细胞极性基因一样潦草 ，导致肿瘤生长的果蝇 ，其中肿瘤细胞也显示侵入行为的突变之间的合作。在这里，一个简单的协议为良性和浸润性肿瘤的诱导和观察提出。
Abstract
果蝇照亮了我们的正常发育和疾病的遗传基础的了解，在过去的几十年，今天它仍然极大地促进我们对复杂疾病的1-7了解。肿瘤从良性到转移状态的进程是一个复杂的事件8，并已在模拟果蝇 ，以帮助我们更好地了解这种疾病9的遗传基础。在这里，我提出了一个简单的协议，以基因诱导，观察并分析肿瘤的果蝇幼虫的进展。肿瘤诱导的技术是基于MARCM系统10上，并利用活化的癌基因，的Ras V12和细胞极性基因丢失（ 乱写 ， 光盘大和致死巨型幼虫 ），以产生浸润性肿瘤9之间的合作。我将演示如何将这些肿瘤可在完整的幼虫进行可视化和那么如何将这些可解剖出作进一步分析。这里介绍的简化协议应该有可能使这种技术通过兴趣了解一个基因在肿瘤侵袭中的作用的调查予以确认。
Introduction
肿瘤从良性到转移状态的进展是明智步骤过程，其特征是存在于主体8的保护机制逃避。例如肿瘤细胞在体内必须能够逃避细胞凋亡和免疫系统，突破了专门的细胞外基质（ECM）称为基底膜，并克服了细胞周围的8所施加的社会控制。它是通过一步明智的进展，癌症细胞获得迁移和殖民遥远的地方在一个称为转移过程的能力。我们如何在肿瘤细胞克服了身体产生的障碍的认识仍处于起步阶段，但是，从研究新兴的画面迄今为止所做指向由癌细胞11-13重复使用正常的发育过程及信号通路。
果蝇果蝇得到了大幅我们UND贡献erstanding通过使用开发了在过去几十年中14-17先进的基因技术正常发育和疾病。使用我们已经到达了一个更好地了解各种癌基因和抑癌基因的18-22诱变和表达的工具。然而，肿瘤转移是一些已被主要研究了细胞培养模型23,24以及各种异种移植模型25-27的遗传病变之间的合作的结果。这些模型虽然厉害有其局限性，因为他们不完全模仿在活的有机体中发现的条件。此外，在现有的小鼠转基因模型是繁琐，不利于侵入行为28,29遗传分析。一些研究试图了解肿瘤细胞的侵袭果蝇 30,31。这些技术主要是利用原发肿瘤移植到主机，然后依靠跟踪TRAnsplanted肿瘤侵犯邻近组织32,33。一个强大的技术，称为MARCM 10适应了Pagliarini和徐肿瘤浸润在果蝇 9建模。肿瘤侵犯的这家优雅的遗传模型利用激活的癌基因和细胞极性的丧失之间的合作。这种造型的优势在于，所述浸润性肿瘤是在一个完整的有机体从而绕过需要组织移植产生的事实。带来的致癌合作，活化的癌基因类似的Ras V12是在细胞中表达的在幼虫眼触角光盘克隆。由于MARCM技术的结果，这些克隆还标有绿色荧光蛋白（GFP），方便的可视化和由合子细胞极性突变体像致命的巨型幼虫 ， 字迹潦草 ，和光盘大，其结果是在绿色荧光蛋白标记的浸润性肿瘤头侧复杂。在这个报告中，我演示如何诱导，并且无论是在一个完整的幼虫的上下文并在解剖出头侧复杂的可视化这些侵入性肿瘤。肿瘤诱导这里提出利用对果蝇第二染色体的试剂。在表2中，我提供股票上，可以被用于相同目的的X和第 3染色体的列表。我认为，这种简化的协议将使这种技术容易接触到感兴趣的研究人员了解肿瘤进展的分子基础。
Protocol
1。诱导良性非侵入性肿瘤使用表2中列出的股票为良性肿瘤的诱导。 准备一个起始培养以下 基因型的“测试仪”股票：Y，W，EY-FLP1;浴池- GAL80，FRT40A; Act5C> Y +> Gal4的，UAS-GFP 准备一个起始培养的“测试”股票下列基因型：W; FRT40A，UAS-V12的Ras / CYO 收集来自“测试仪”股票10只雌性童女和十男性从“测试”的股票。 放置他…
Representative Results
作为协议的结果，在这里呈现的用户将能够通过过度激活的癌基因在幼虫眼触角成虫盘诱发良性肿瘤。用户也将能够通过过表达活化的癌基因在细胞的克隆以诱导侵入性肿瘤在眼触角光盘也突变体的细胞极性基因。肿瘤可以很容易地可视化，用荧光显微镜在整个幼虫或在已解剖出的幼虫体腔（ 图1）的头部复合物“绿色荧光”组织的帮助。良性肿瘤将被局限在眼睛触角盘复杂的GFP阳性…
Discussion
癌症是一种复杂的疾病有一个更好的了解，今天比过去。然而，仍有许多需要学习和解释之前，我们并不拥有相关机制的全貌。这里提出的简单的协议，能够诱导基因在整个生物体良性和侵入性肿瘤，然后研究与肿瘤的此模型中的进展有关的生物学。大部分在果蝇和其他生物的现有技术或者利用基于细胞的培养系统，以了解肿瘤发生及转移，或一个系统，它采用原发肿瘤组织移植入成年的?…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
研究在我的实验室的支持，生物学启动资金的WKU部，WKU研究基金会RCAP，我承认＃11-8032，并通过从全国学院普通医学科学研究所父母捐款资助，KBRIN-AREA补助生在奖号5P20GM103436-13。我还要感谢田旭中博士实验室的我被介绍给这个技术和雷蒙德Pagliarini博士谁首先建立这种技术在许实验室。
Materials
| 10X PBS (phosphate buffered saline) pH 7.2 stock solution | Invitrogen, Sigma Aldrich | ||
| Chilled 1X PBS pH7.2 working solution | Invitrogen, Sigma Aldrich | Make fresh and refrigerate, can be used up to a week | |
| Flynap | Carolina Biologicals | Fly anesthesia needed to anesthetize larvae | |
| Fixative | 0.1M PIPES, pH 7.2, 4% Paraformaldehyde | Needed to fix the dissected cephalic complex | |
| Ice Bucket | Several | Maintain solutions on ice. Also, dissect cephalic complex in chilled 1X PBS and then place on ice in an Eppendorf tube | |
| 1.7ml Eppendorf tube | Various | ||
| Glass slides, cover glass | Fisher Scientific | ||
| Vectashield Mounting Media or any other mounting media | Vector Laboratories | ||
| Halocarbon 200 or 700 Oil | Polysciences Inc. or Halocarbon.com | Halocarbon 200 is used to mount the larvae for visualization on a fluorescence stereoscope | |
| Sally Hansen “Hard as Nails” nail polish | Can be found at any general merchandise store | Needed to seal the edges of Coverslip | |
| A Leica MZ16.5 fluorescence stereomicroscope or any other fluorescence stereomicroscope | Leica and others | Needed to observe the GFP fluorescence in larvae | |
| Dumont #5 forceps | Fine Science Tools | ||
| Pyrex 9 well spot plate or any other dissection dish | Sigma Aldrich | ||
| Paint Brush | Can be found at any general merchandise store | ||
| Table 1. Materials needed to perform the experimental protocol presented in this article. |
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Citazione di questo articolo
Srivastava, A. A Protocol for Genetic Induction and Visualization of Benign and Invasive Tumors in Cephalic Complexes of Drosophila melanogaster. J. Vis. Exp. (79), e50624, doi:10.3791/50624 (2013).