Name: injecting gryllus bimaculatus eggs
Uploaded: 2019-08-22T13:00:00
Description: Watch this Scientific Journal Video about Injecting Gryllus bimaculatus Eggs at JoVE.com

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。

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。
<ol><li>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。</li>
	<li>放置一个3轴微操作器，将注射针操纵到位（图1A）。</li>
	<l...

<ol>
	<li>Abzhanov, A., 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=Are+we+there+yet?+Tracking+the+development+of+new+model+systems.">Are we there yet? Tracking the development of new model systems.</a> Trends in Genetics. 24 (7), 353-360 (2008).</li><li>Krebs, H. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+August+Krogh+principle:+&#8220;For+many+problems+there+is+an+animal+on+which+it+can+be+most+conveniently+studied.">The August Krogh principle: &#8220;For many problems there is an animal on which it can be most conveniently studied.</a> Journal of Experimental Zoology Part B: Molecular and Developmental Evolution. 194 (1), 221-226 (1975).</li><li>Krogh, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Progress+of+Physiology.">The Progress of Physiology.</a> American Journal of Physiology. 90 (2), 243-251 (1929).</li><li>Huber, F., Moore, T. E., Loher, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Cricket neurobiology and behavior. , (1989).</li><li>Horch, H. W., Mito, T., Popadic, A., Ohuchi, H., Noji, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The Cricket as a Model Organism: Development, Regeneration, and Behavior. , (2017).</li><li>Misof, B., 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=Phylogenomics+resolves+the+timing+and+pattern+of+insect+evolution.">Phylogenomics resolves the timing and pattern of insect evolution.</a> Science. 346 (6210), 763-767 (2014).</li><li>Nakamura, T., 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=Imaging+of+transgenic+cricket+embryos+reveals+cell+movements+consistent+with+a+syncytial+patterning+mechanism.">Imaging of transgenic cricket embryos reveals cell movements consistent with a syncytial patterning mechanism.</a> Current Biology. 20 (18), 1641-1647 (2010).</li><li>Shinmyo, Y., 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=piggyBac-mediated+somatic+transformation+of+the+two-spotted+cricket,+Gryllus+bimaculatus.">piggyBac-mediated somatic transformation of the two-spotted cricket, Gryllus bimaculatus.</a> Development, growth &amp; differentiation. 46 (4), 343-349 (2004).</li><li>Watanabe, T., 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=Non-transgenic+genome+modifications+in+a+hemimetabolous+insect+using+zinc-finger+and+TAL+effector+nucleases.">Non-transgenic genome modifications in a hemimetabolous insect using zinc-finger and TAL effector nucleases.</a> Nature communications. 3, 1017 (2012).</li><li>Wang, H., La Russa, M., Qi, L. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CRISPR/Cas9+in+Genome+Editing+and+Beyond.">CRISPR/Cas9 in Genome Editing and Beyond.</a> Annual Review of Biochemistry. 85 (1), 227-264 (2016).</li><li>Awata, H., Watanabe, T., Hamanaka, Y., Mito, T., Noji, S., Mizunami, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Knockout+crickets+for+the+study+of+learning+and+memory:+Dopamine+receptor+Dop1+mediates+aversive+but+not+appetitive+reinforcement+in+crickets.">Knockout crickets for the study of learning and memory: Dopamine receptor Dop1 mediates aversive but not appetitive reinforcement in crickets.</a> Scientific Reports. 5, 15885 (2015).</li><li>Horch, H. W., Liu, J. J., Mito, T., Popadic, A., Watanabe, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protocols+in+the+Cricket.">Protocols in the Cricket.</a> The Cricket as a Model Organism: Development, Regeneration, and Behavior. , 327-370 (2017).</li><li>Watanabe, T., Noji, S., Mito, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome+Editing+in+the+Cricket,+Gryllus+bimaculatus.">Genome Editing in the Cricket, Gryllus bimaculatus.</a> Genome Editing in Animals. , 219-233 (2017).</li><li>Kainz, F., Ewen-Campen, B., Akam, M., Extavour, C. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Notch/Delta+signalling+is+not+required+for+segment+generation+in+the+basally+branching+insect+Gryllus+bimaculatus.">Notch/Delta signalling is not required for segment generation in the basally branching insect Gryllus bimaculatus.</a> Development. 138 (22), 5015-5026 (2011).</li><li>Miyawaki, K., 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=Involvement+of+Wingless/Armadillo+signaling+in+the+posterior+sequential+segmentation+in+the+cricket,+Gryllus+bimaculatus+(Orthoptera),+as+revealed+by+RNAi+analysis.">Involvement of Wingless/Armadillo signaling in the posterior sequential segmentation in the cricket, Gryllus bimaculatus (Orthoptera), as revealed by RNAi analysis.</a> Mechanisms of Development. 121 (2), 119-130 (2004).</li><li>Donoughe, S., Kim, C., Extavour, C. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-throughput+live-imaging+of+embryos+in+microwell+arrays+using+a+modular+specimen+mounting+system.">High-throughput live-imaging of embryos in microwell arrays using a modular specimen mounting system.</a> Biology Open. 7 (7), bio031260 (2018).</li><li>Donoughe, S., Nakamura, T., Ewen-Campen, B., Green, D. A., Henderson, L., Extavour, C. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=BMP+signaling+is+required+for+the+generation+of+primordial+germ+cells+in+an+insect.">BMP signaling is required for the generation of primordial germ cells in an insect.</a> Proceeding of the National Academy of Science USA. 111 (11), 4133-4138 (2014).</li><li>Larson, E., Andres, J., Harrison, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Influence+of+the+male+ejaculate+on+post-mating+prezygotic+barriers+in+field+crickets.">Influence of the male ejaculate on post-mating prezygotic barriers in field crickets.</a> PLOS ONE. 7 (10), e46202 (2012).</li><li>Donoughe, S., Extavour, C. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Embryonic+development+of+the+cricket+Gryllus+bimaculatus.">Embryonic development of the cricket Gryllus bimaculatus.</a> Developmental Biology. , (2016).</li><li>Matsuoka, Y., 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=Short+germ+insects+utilize+both+the+ancestral+and+derived+mode+of+Polycomb+group-mediated+epigenetic+silencing+of+Hox+genes.">Short germ insects utilize both the ancestral and derived mode of Polycomb group-mediated epigenetic silencing of Hox genes.</a> Biology Open. 4 (6), 702-709 (2015).</li><li>Rosenberg, M., Lynch, J., Desplan, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heads+and+tails:+Evolution+of+antero-posterior+patterning+in+insects.">Heads and tails: Evolution of antero-posterior patterning in insects.</a> Biochimica et biophysica acta. 1789 (4), 333-342 (2009).</li><li>Bacon, J., Strausfeld, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nonrandom+resolution+of+neuron+arrangements.">Nonrandom resolution of neuron arrangements.</a> Neuroanatomical Techniques: Insect Nervous System. , 357-372 (1980).</li></ol>

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?...

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。

<table>
	<tbody>
		<tr>
			<td>Fluorescent dissecting microscope</td>
			<td>Leica</td>
			<td>M165 FC</td>
			<td>Stereomicroscope with fluorescence</td>
			<td></td>
		</tr>
		<tr>
			<td>External light source for fluorescence</td>
			<td>Leica</td>
			<td>EL 6000</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Microinjector</td>
			<td>Narishige</td>
			<td>IM-300</td>
			<td>-Accessories may include Injection Needles Holder, Input Hose (with a hose connector), AC Power Cord, Foot Switch, Silicone Rubber Gasket-</td>
			<td></td>
		</tr>
		<tr>
			<td>mCherry filter cube</td>
			<td>Leica</td>
			<td>M205FA/M165FC</td>
			<td>Filter cube for mCherry or similar red dye will work</td>
			<td></td>
		</tr>
		<tr>
			<td>Micromanipulator</td>
			<td>World Precision Instruments, Inc.</td>
			<td>M3301R</td>
			<td>Used with Magnetic Stand (Narishige, Type GJ-8)</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnetic stand</td>
			<td>Narishige</td>
			<td>MMO-202ND</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Pipette Holder (Needle holder)</td>
			<td>Narishige</td>
			<td>HD-21</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td colspan="2">Tubing to connect air source to microinjector</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Egg well stamp</td>
			<td>3D printed</td>
			<td>custom</td>
			<td>3D printed on a Lulzbot Taz 5 using Poly Lactic Acid thermoplastic</td>
			<td></td>
		</tr>
		<tr>
			<td>Microwave</td>
			<td>various</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Incubator or temperature controlled room</td>
			<td>various</td>
			<td></td>
			<td>Temperatures of 23.5-26&#176;C are needed.</td>
			<td></td>
		</tr>
		<tr>
			<td>cricket food</td>
			<td>various</td>
			<td></td>
			<td>cat food or fish flakes are appropriate food.&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>cricket wter</td>
			<td>vairous</td>
			<td></td>
			<td>Water can be held in vials and presented to crickets through cotton balls</td>
			<td></td>
		</tr>
		<tr>
			<td>cricket shelter</td>
			<td>arious</td>
			<td></td>
			<td>Shelter materials can include crumpled paper towels or egg cartons</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass capillary tubes</td>
			<td>World Precision Instruments, Inc.</td>
			<td>Item no. 1B100F-4</td>
			<td>Kwik-Fil&#8482; Borosilicate Glass Capillaries, 100mm length, 0.58 mm ID, 1.0 mm OD, with filament</td>
			<td></td>
		</tr>
		<tr>
			<td>Micropipette puller</td>
			<td>Flaming/Brown</td>
			<td>Model P-97</td>
			<td>Distributed by Sutter Instrument Co.</td>
			<td></td>
		</tr>
		<tr>
			<td>Beveller/Micro grinder</td>
			<td>Narishige</td>
			<td>Model EG-45/EG-400</td>
			<td>EG-400 includes a microscope head</td>
			<td></td>
		</tr>
		<tr>
			<td>Petri dishes</td>
			<td>CellTreat</td>
			<td>Product code 229693</td>
			<td>90mm diameter</td>
			<td></td>
		</tr>
		<tr>
			<td>Play Sand</td>
			<td>Sandtastik Products Ltd.</td>
			<td>B003U6QLVS</td>
			<td>White play sand</td>
			<td></td>
		</tr>
		<tr>
			<td>Agarose</td>
			<td>American Bioanalytical</td>
			<td>AB000972</td>
			<td>Agarose GPG/LE ultrapure</td>
			<td></td>
		</tr>
		<tr>
			<td>Egg Strainer: Extra Fine Twill Mesh Stainless Steel Conical Strainers</td>
			<td>US Kitchen Supply</td>
			<td>Model SS-C123</td>
			<td>Pore size should be between 0.5 - 1.0 mm</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin Streptomycin</td>
			<td>Gibco by Life Technologies</td>
			<td>Ref 15070-063</td>
			<td>Pen Strep</td>
			<td></td>
		</tr>
		<tr>
			<td>Plastic tweezers</td>
			<td>Sipel Electronic SA</td>
			<td>P3C-STD</td>
			<td>Black Static Dissipative, 118mm</td>
			<td></td>
		</tr>
		<tr>
			<td>syringe filters, 25mm diameter, 0.45 &#181;m</td>
			<td>Nalgene</td>
			<td>725-2545</td>
			<td>Use with 1 ml syringe</td>
			<td></td>
		</tr>
		<tr>
			<td>1 mL syringe, with Tuberculin Slip Tip</td>
			<td>Becton Dickinson</td>
			<td>309602</td>
			<td>Use with syring filter to filter Injection Buffer , Luer-Lok tip syringes would also work</td>
			<td></td>
		</tr>
		<tr>
			<td>Air tank (optional)</td>
			<td>Midwest Products</td>
			<td>Air Works&#174;</td>
			<td>Portable air tank</td>
			<td></td>
		</tr>
		<tr>
			<td rowspan="2">Rhodamine dye</td>
			<td rowspan="2">Thermofisher</td>
			<td rowspan="2">D-1817</td>
			<td rowspan="2">dextran, tetramethylrhodamine 10,000MW,</td>
			<td></td>
		</tr>
		<tr>
			<td>20 mL loading tips</td>
			<td>Eppendorf</td>
			<td>Order no. 5242 956.003</td>
			<td>epT.I.P.S. 20uL Microloader</td>
			<td></td>
		</tr>
		<tr>
			<td>Compound microscope</td>
			<td>Zeiss</td>
			<td>Axioskope 2 plus</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>20X objective</td>
			<td>Ziess</td>
			<td>Plan-Apochromat 20x/0.75 M27</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>camera</td>
			<td>Leica</td>
			<td>DMC 5400</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Leica Application Suite&#160; software</td>
			<td>Leica</td>
			<td>LAS</td>
			<td>Version 4.6.2 used here</td>
			<td></td>
		</tr>
	</tbody>
</table>

injecting gryllus bimaculatus eggs

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。

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%?...

Watch this Scientific Journal Video about Injecting Gryllus bimaculatus Eggs at JoVE.com

Injecting Gryllus bimaculatus Eggs

注射格里鲁斯双形蛋

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。

Altering gene function in a developing organism is central to different kinds of experiments. While tremendously powerful genetic tools have been ...

该技术是用于测定板球胚胎发育或幼虫发育的实验基础方法。该技术的主要优点是，它具有灵活性，将支持几种类型的实验方法，如使用RNA干扰或基因组操作的方法。实验室的主要调查员哈德利·威尔逊·霍奇将展示这一程序。要开始此过程，用滴水将斜面的旋转研磨机平面弄湿。将拉针放入斜面，将其转动到 20 度角。放下针头，直到它只接触研磨板，并斜面两到三分钟。通过将斜面针放在已粘附在玻璃显微镜幻灯片上的双斗胶带上来评估斜面。将幻灯片放在配备相机和图像采集软件的复合显微镜的舞台上。使用 20 倍目标获取针头图像。使用成像软件测量针头的内部流明直径，刚到斜面开口。丢弃开口小于 8 微米或大于 12 微米的针头。首先，在水中制作40毫升1%的加糖，倒入10厘米的培养皿中。在蛋井凝固之前，先在蛋井表面盖上蛋井盖章。凝固一次，取出印戳以露出井口。接下来，用含有1%青霉素链霉素的HBS填充阿加罗斯井板。将盖子放在盘子上，用 Parafilm 包装，并将其存放在摄氏四度。在此之后，用白色游乐场沙子填充一个35毫米的培养皿，制作一个鸡蛋收集盘，让板球人产卵。用纸巾方形覆盖鸡蛋盘，切入约18由18厘米。用纸巾加上自来水。然后，倾斜培养皿，轻轻挤压顶部以去除多余的水。将纸巾方的角落塞到盘子下面，将鸡蛋放在倒置的盖子中，这将有助于将纸巾放在原位。将鸡蛋盘放入板球箱，让成年女性将鸡蛋放入一到两个小时。同时，让加糖盘加热到室温，准备将鸡蛋放在注射中。准备好后，从板球箱中取出现在含有刚产的鸡蛋的鸡蛋收集盘。取下纸巾，将孔径介于 0.5 至 1 毫米之间的过滤器放在烧杯上。在轻轻的自来水下将产卵盘的内容冲洗到过滤器中。沙粒会通过过滤器网落入下面的烧杯，而板球蛋留在过滤器篮中。用反渗透水填充容器，并放在托盘中。将过滤器倒置在容器上，用水龙头敲打盘子，将鸡蛋倒入水中。鸡蛋会沉入容器的底部。接下来，用剪刀将 P1000 移液器尖端切开，使开口直径约为 3 毫米。将这个尖端放在P1000移管器上，并用它来将鸡蛋从容器转移到阿加罗斯蛋模盘上。使用塑料钳子，将鸡蛋排在加糖井中。每个鸡蛋都会沉入单个井的底部。用盖子盖住培养皿，直到准备注射。首先，将鸡蛋盘放在解剖显微镜下，并选择大约10倍的低放大倍率。使用 20 微升加载尖端和 P10 移液器绘制 1.5 微升喷射溶液，并将加载尖端插入注射针的宽端。将注射溶液排出注射针中。将针头插入注射壳体并拧紧，确保针头正确且牢固地插入壳体中。然后小心地将注射壳体插入微操纵器中，小心不要被针头打断或受伤。通过显微镜观察鸡蛋和针头时，将针头移近菜盘网格左上角的 X。放下针头，直到尖端进入HBS加上青霉素链霉素缓冲溶液在菜中。将针头放在视野中，移动蛋盘，使针头比鸡蛋更接近菜的边缘几毫米。在此之后，将显微镜设置为适合罗达明的过滤器，以观察针头中的荧光并聚焦在针头上。在微注射器上，顺时针缓慢转动平衡旋钮，直到注射溶液开始从针头中泄漏到悬浮液中。然后稍微逆时针转动旋钮，直到染料停止从针头中泄漏。在针仍然以视场为中心时，移动蛋盘，使针头瞄准要先注射的鸡蛋。将放大倍数调整到大约 50 倍，以便单个鸡蛋填充大部分视野。使用微操纵器和手放在鸡蛋盘上，将针头移动到注射位置。使用微操纵器推进针头，并将尖端插入要注射的第一个卵子中，确保将针头插入卵子长度的 20% 至 30%，从卵子的后端垂直于卵子的长轴。将溶液与喷射脚踏板或喷射按钮一起注入微注射器。鸡蛋内一小块荧光材料表明注射成功。在此之后，从鸡蛋中收回针头。如果鸡蛋在缩回针头时无意中从井中抬出，则使用小钳子将鸡蛋推回井中，同时缩回针头。在这项研究中，板球卵子被注射使用一种技术，作为许多实验中的基础方法，包括但不限于RNA干扰和基因组操作。存活率是评价实验成功与否的重要参数。大多数死蛋很容易被视觉识别，因为蛋黄和胚胎组织开始不均匀地团块，最终大部分蛋黄会迁移到蛋的一侧。在四到六天后评估时，超过85%的未注射卵子存活下来，而注射实验试剂的卵子的存活率通常较低。为了了解尝试的实验操作的真正效果，需要控制注射本身的所有方面，包括针刺、染料和缓冲液的引入，或车辆的存在或双绞合RNA。评估注射的表型结果的方式取决于注射的表型结果。例如，由于特定的 mRNA 击倒而导致的表型变化在总解剖水平上可能很明显。另一方面，如果使用背负转位酶将编码EGFP标记组蛋白2B基因的cDNA插入板球基因组，则利用荧光来激发EGFP标记的组蛋白2B蛋白。调整平衡，使针头不堵塞是此过程的关键步骤。由于蛋黄有时会堵塞针尖，因此您可能需要在随后的注射后进行几次调整。板球是一种血小昆虫，其基础分支，以研究良好的全脂代谢昆虫，如果蝇。研究板球的发展将有助于我们了解整个动物王国的进化和发展。这种技术需要一些练习。我们建议练习控制注射，直到注射后四五天的存活率至少为 80%

Research

JoVE Journal

Developmental Biology

Injecting Gryllus bimaculatus Eggs

Salinity-dependent Toxicity Assay of Silver Nanocolloids Using Medaka Eggs

白银纳米胶体的盐度依赖性毒力测定使用青鳉鸡蛋

Salinity is an important characteristic of the aquatic environment. For aquatic organisms it defines the habitats of freshwater, brackish water, and ...

科研

发育生物学

In Ovo Feeding of Commercial Broiler Eggs: An Accurate and Reproducible Method to Affect Muscle Development and Growth

In Ovo Feeding of Commercial Broiler Eggs: An Accurate and Reproducible Method to Affect Muscle Development and Growth

在 奥沃 商业肉鸡蛋的喂养：一种影响肌肉发育和生长的准确且可重复的方法

Within the past three decades, red meat and poultry scientists focused on developing strategies and technologies to manipulate muscle development during ...

Investigating Development with Optogenetic Signaling Manipulation in Zebrafish

Optogenetic Signaling Activation in Zebrafish Embryos

Manipulating Signaling in Zebrafish Embryos to Decode Cell Fate Decisions (Video) | JoVE

斑马鱼胚胎中的光遗传学信号激活

Signaling pathways orchestrate fundamental biological processes, including development, regeneration, homeostasis, and disease. Methods to experimentally ...

Enhancing Protein Visualization with Antibody-Mediated Live Imaging

Visualizing Low-Abundance Proteins and Post-Translational Modifications in Living Drosophila Embryos via Fluorescent Antibody Injection

Unveiling the Dynamics of Mechanical and Biochemical Signals in Animal Morphogenesis Research (Video) | JoVE

通过荧光抗体注射观察活果蝇胚胎中的低丰度蛋白质和翻译后修饰

Visualization of proteins in living cells using GFP (Green Fluorescent Protein) and other fluorescent tags has greatly improved understanding of protein ...