注射格里鲁斯双形蛋
1Colby College, 2Division of Evolutionary Developmental Biology,National Institute for Basic Biology, 3Department of Biological Sciences,National University of Singapore, 4Department Biology and Department of Neuroscience,Bowdoin College, 5Department of Organismic and Evolutionary Biology and Department of Molecular and Cellular Biology,Harvard University
Summary
在这里，我们提出了一个协议，注射板球卵，该技术作为基础方法在许多实验在板球，包括但不限于，RNA干扰和基因组操作。
Abstract
改变发育中的生物体的基因功能是不同实验的核心。虽然在传统的模型系统中已经开发出了非常强大的遗传工具，但在大多数其他生物体中很难操纵基因或信使RNA（mRNA）。同时，进化和比较方法依赖于对许多不同的物种基因功能的探索，因此需要开发和调整目前遗传性可操作的表达之外的表达技术。物种。该协议描述了一种将试剂注射到板球卵子中的方法，以测定给定操作对胚胎或幼虫发育的影响。介绍了如何用尖刺针头收集和注射卵子的说明。这种相对简单的技术是灵活的，并可能适应其他昆虫。一个实验可以收集和注射几十个卵子，仅缓冲注射的存活率随着实践而提高，可以高达80%。该技术将支持几种实验方法，包括注射药理剂，体外封顶mRNA来表达感兴趣的基因，双链RNA（dsRNA）实现RNA干扰，定期使用聚集与CRISPR相关蛋白9（Cas9）试剂配合进行间隔短回溯重复（CRISPR）进行基因组修饰，并可转位元素生成瞬态或稳定的转基因线。
Introduction
改变生物体中的基因组或影响基因表达的能力是设计多种实验测试功能因果关系的基础。在传统基因实验室动物模型系统（如Musmusculus、Danio）之外的生物体中，基因组和非基因组修饰技术也对于比较和进化相关工作也至关重要。 雷里奥、德罗索菲拉·梅拉诺加斯特和卡埃诺哈布迪西·埃莱甘斯。无论是理解有机体多样性1的愿望，还是对Krogh原则的坚持，对于每一个生物问题，都有一个最适合其解决方案2、3、修改基因组或影响基因表达是现代实验设计的关键。
板球Gryllus双形球是一个新兴的模型系统。用于上个世纪的神经学实验4，在过去的二十年里见证了对板球的实验兴趣增加，特别是专注于这种生物体的进化和发展5。板球是一种截带性昆虫，它向经过良好研究的全息昆虫（如D.黑色素气和三硼酸酯6）分枝。由于其在进化树上的有用位置，科学家们有兴趣在这种昆虫中提出现代的、复杂的实验问题，这导致人们越来越有兴趣调整分子工具，用于G.双形虫。
将分子试剂注入板球卵子可用于基因组修饰实验以及胚胎中基因表达的非基因组操作。例如，转基因G.双体细胞携带eGFP插入已经创建使用转座酶piggyBac7，8。研究者已经成功地利用锌指核酸酶（ZFNs）和转录激活器样（TAL）效应素（TALENs）在特定的基因组区域9中引入双链断裂。虽然ZFN和TALEN允许在四大模型系统之外的动物进行特定定位，但这些试剂已被CRISPR/Cas9系统迅速超越，该系统使用更简单、效率更高、灵活性高。CRISPR已用于G.双核糖核酸生产敲出11以及敲入线12，13 除了基因组修饰，dsRNA可以注射到鸡蛋中，以敲落mRNA表达在开发胚胎，使调查人员了解特定记录的作用在整个开发14，15。一些关于如何注射板球蛋的有限细节已经发表于12。
在这里，我们描述了注射早期G.双形卵子的详细方案。该协议是有效的，很容易适应各种实验室设置，注射材料，并可能对其他昆虫。虽然设计和实施基因组修饰和击倒实验的其他细节已经在其他地方发表，这些方法最终将依赖于此处详述的注射方案。
Protocol
1. 硬件设置和材料准备 注：有关解决方案、试剂和设备详细信息的准备，请参阅表 1和材料表。 设置解剖显微镜，以便看到鸡蛋并引导注射针。（图1A显示了装有荧光的解剖显微镜。荧光能力是有利的，但不是必须的。 放置一个3轴微操作器，将注射针操纵到位（图1A）。 <l…
Representative Results
板球很容易在潮湿的材料中产卵，并提供足够的材料，如潮湿的沙或污垢，诱导他们下大量的鸡蛋。如果首先在洁净的沙中产卵8~10小时，这尤其有效。解剖显微镜、显微注射器和微操作器（图1A）可用于向单个卵子注入各种材料（图1D）。鸡蛋在后端附近被注射，沿前后轴大约20-30%?…
Discussion
该技术的两个主要挑战是最佳针尺寸和生存能力的相关问题。虽然较小的针头可以提高生存能力，但流明较窄的针头在工作中具有更大程度的毛细管力，这使得蛋黄更有可能进入针头，导致其堵塞。在最好的情况下，只需注射另一个卵子或清除针头即可清除堵塞，如上所述。也可以尝试增加显微注射器上的平衡压力，直到蛋黄被推出针头。这项技术的另一个主要挑战是生存能力。在我们手中，注?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
本项目报告的研究得到了国家卫生研究院国家普通医学研究所颁发的机构发展奖（IDeA）的支持，授予编号为P20GM10342至HH3，以及NSF奖号IOS-1257217Cge。
Materials
| Fluorescent dissecting microscope | Leica | M165 FC | Stereomicroscope with fluorescence | |
| External light source for fluorescence | Leica | EL 6000 | ||
| Microinjector | Narishige | IM-300 | -Accessories may include Injection Needles Holder, Input Hose (with a hose connector), AC Power Cord, Foot Switch, Silicone Rubber Gasket- | |
| mCherry filter cube | Leica | M205FA/M165FC | Filter cube for mCherry or similar red dye will work | |
| Micromanipulator | World Precision Instruments, Inc. | M3301R | Used with Magnetic Stand (Narishige, Type GJ-8) | |
| Magnetic stand | Narishige | MMO-202ND | ||
| Pipette Holder (Needle holder) | Narishige | HD-21 | ||
| Tubing to connect air source to microinjector | ||||
| Egg well stamp | 3D printed | custom | 3D printed on a Lulzbot Taz 5 using Poly Lactic Acid thermoplastic | |
| Microwave | various | |||
| Incubator or temperature controlled room | various | Temperatures of 23.5-26°C are needed. | ||
| cricket food | various | cat food or fish flakes are appropriate food. | ||
| cricket wter | vairous | Water can be held in vials and presented to crickets through cotton balls | ||
| cricket shelter | arious | Shelter materials can include crumpled paper towels or egg cartons | ||
| Glass capillary tubes | World Precision Instruments, Inc. | Item no. 1B100F-4 | Kwik-Fil™ Borosilicate Glass Capillaries, 100mm length, 0.58 mm ID, 1.0 mm OD, with filament | |
| Micropipette puller | Flaming/Brown | Model P-97 | Distributed by Sutter Instrument Co. | |
| Beveller/Micro grinder | Narishige | Model EG-45/EG-400 | EG-400 includes a microscope head | |
| Petri dishes | CellTreat | Product code 229693 | 90mm diameter | |
| Play Sand | Sandtastik Products Ltd. | B003U6QLVS | White play sand | |
| Agarose | American Bioanalytical | AB000972 | Agarose GPG/LE ultrapure | |
| Egg Strainer: Extra Fine Twill Mesh Stainless Steel Conical Strainers | US Kitchen Supply | Model SS-C123 | Pore size should be between 0.5 – 1.0 mm | |
| Penicillin Streptomycin | Gibco by Life Technologies | Ref 15070-063 | Pen Strep | |
| Plastic tweezers | Sipel Electronic SA | P3C-STD | Black Static Dissipative, 118mm | |
| syringe filters, 25mm diameter, 0.45 µm | Nalgene | 725-2545 | Use with 1 ml syringe | |
| 1 mL syringe, with Tuberculin Slip Tip | Becton Dickinson | 309602 | Use with syring filter to filter Injection Buffer , Luer-Lok tip syringes would also work | |
| Air tank (optional) | Midwest Products | Air Works® | Portable air tank | |
| Rhodamine dye | Thermofisher | D-1817 | dextran, tetramethylrhodamine 10,000MW, | |
| 20 mL loading tips | Eppendorf | Order no. 5242 956.003 | epT.I.P.S. 20uL Microloader | |
| Compound microscope | Zeiss | Axioskope 2 plus | ||
| 20X objective | Ziess | Plan-Apochromat 20x/0.75 M27 | ||
| camera | Leica | DMC 5400 | ||
| Leica Application Suite software | Leica | LAS | Version 4.6.2 used here | |
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Cite This Article
Barry, S. K., Nakamura, T., Matsuoka, Y., Straub, C., Horch, H. W., Extavour, C. G. Injecting Gryllus bimaculatus Eggs. J. Vis. Exp. (150), e59726, doi:10.3791/59726 (2019).