Name: egg microinjection and efficient mating for genome editing in the firebrat thermobia domestica
Uploaded: 2020-10-20T15:00:00
Description: Watch this Scientific Journal Video about Egg Microinjection and Efficient Mating for Genome Editing in the Firebrat Thermobia domestica at JoVE.com

TO 和 TD 分别得到 JSPS KAKENHI 赠款编号 19H02970 和 20H02999 的支持。

1. 实验室菌落的维护
<ol><li>为了维持野生类型和突变种群，使用大型塑料容器（460 毫米 x 360 mm x 170 mm）与常规人工鱼食、顶部有通风孔的塑料杯中的水、用于隐藏昆虫的折叠纸和用于产卵的分层棉花（图 2A）。将所有 T. 家用 文化保留在 37 °C 孵化器内，并将每个容器内的相对湿度 （RH） 设置为 60%-80%。 注：由于 T.家政 从大气中吸收水蒸气11?...

<ol>
	<li>Peterson, M. D., Rogers, B. T., Popadi&#263;, A., Kaufman, T. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+embryonic+expression+pattern+of+labial,+posterior+homeotic+complex+genes+and+the+teashirt+homologue+in+an+apterygote+insect.">The embryonic expression pattern of labial, posterior homeotic complex genes and the teashirt homologue in an apterygote insect.</a> Development Genes and Evolution. 209, 77-90 (1999).</li><li>Farris, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Developmental+organization+of+the+mushroom+bodies+of+Thermobia+domestica+(Zygentoma,+Lepismatidae):+Insights+into+mushroom+body+evolution+from+a+basal+insect.">Developmental organization of the mushroom bodies of Thermobia domestica (Zygentoma, Lepismatidae): Insights into mushroom body evolution from a basal insect.</a> Evolution and Development. 7, 150-159 (2005).</li><li>Kamae, Y., Tanaka, F., Tomioka, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+cloning+and+functional+analysis+of+the+clock+genes,+Clock+and+cycle,+in+the+firebrat+Thermobia+domestica.">Molecular cloning and functional analysis of the clock genes, Clock and cycle, in the firebrat Thermobia domestica.</a> Journal of Insect Physiology. 56, 1291-1299 (2010).</li><li>Ohde, T., Masumoto, M., Yaginuma, T., Niimi, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Embryonic+RNAi+analysis+in+the+firebrat,+Thermobia+domestica:+Distal-less+is+required+to+form+caudal+filament.">Embryonic RNAi analysis in the firebrat, Thermobia domestica: Distal-less is required to form caudal filament.</a> Journal of Insect Biotechnology and Sericology. 105, 99-105 (2009).</li><li>Ohde, T., Yaginuma, T., Niimi, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nymphal+RNAi+analysis+reveals+novel+function+of+scalloped+in+antenna,+cercus+and+caudal+filament+formation+in+the+firebrat,+Thermobia+domestica.">Nymphal RNAi analysis reveals novel function of scalloped in antenna, cercus and caudal filament formation in the firebrat, Thermobia domestica.</a> Journal of Insect Biotechnology and Sericology. 108, 101-108 (2012).</li><li>Bell&#233;s, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Beyond+Drosophila:+RNAi+in+vivo+and+functional+genomics+in+insects.">Beyond Drosophila: RNAi in vivo and functional genomics in insects.</a> Annual Review of Entomology. 55, 111-128 (2010).</li><li>Ohde, T., Takehana, Y., Shiotsuki, T., Niimi, T. <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-based+heritable+targeted+mutagenesis+in+Thermobia+domestica:+A+genetic+tool+in+an+apterygote+development+model+of+wing+evolution.">CRISPR/Cas9-based heritable targeted mutagenesis in Thermobia domestica: A genetic tool in an apterygote development model of wing evolution.</a> Arthropod Structure and Development. 47, 362-369 (2018).</li><li>Nji, C., 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=CRISPR/Cas9+in+insects:+Applications,+best+practices+and+biosafety+concerns.">CRISPR/Cas9 in insects: Applications, best practices and biosafety concerns.</a> Journal of Insect Physiology. 98, 245-257 (2017).</li><li>Brand, P., 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=The+origin+of+the+odorant+receptor+gene+family+in+insects.">The origin of the odorant receptor gene family in insects.</a> eLife. 7, 1-13 (2018).</li><li>Noble-Nesbitt, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Water+balance+in+the+firebrat,+Thermobia+domestica+(Packard).+Exchanges+of+water+with+the+atmosphere.">Water balance in the firebrat, Thermobia domestica (Packard). Exchanges of water with the atmosphere.</a> Journal of Experimental Biology. 50, 745-769 (1969).</li><li>Sweetman, H. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Physical+ecology+of+the+firebrat,+Thermobia+domestica+(Packard).">Physical ecology of the firebrat, Thermobia domestica (Packard).</a> Ecological Monographs. 8, 285-311 (1938).</li><li>Nijhout, H. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Insect Hormones. , (1994).</li><li>Chen, 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=Efficient+detection,+quantification+and+enrichment+of+subtle+allelic+alterations.">Efficient detection, quantification and enrichment of subtle allelic alterations.</a> DNA Research. 19, 423-433 (2012).</li><li>Mashal, R. D., Koontz, J., Sklar, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+of+mutations+by+cleavage+of+DNA+heteroduplexes+with+bacteriophage+resolvases.">Detection of mutations by cleavage of DNA heteroduplexes with bacteriophage resolvases.</a> Nature Genetics. 9, 177-183 (1995).</li><li>Adams, 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=Biological+notes+upon+the+firebrat,+Thermobia+domestica+Packard.">Biological notes upon the firebrat, Thermobia domestica Packard.</a> Journal of the New York Entomological Society. 41, 557-562 (1933).</li></ol>

对于成功生成所需的 T.家 用突变体CRISPR/Cas9，首先重要的是收集足够数量的分阶段胚胎进行注射。对于足够数量的 T.家蛋 的不断收集，关键是选择一个适当的大小的容器，以具有较低的人口密度，因为它将有助于成功完成一系列复杂的交配行为，这是重复后，每个成人摩尔13。男性 T.家庭 成人通过精子间接地将其精子转移给女性。斯威特曼（1938年）报告说?...

热比娅家居属于最基础的昆虫之一，Zygentoma，它保留了祖先的代谢和无翼生命周期。这种基底植物遗传位置和祖先特征为研究地球上昆虫成功背后的机制树立了有吸引力的模型，昆虫覆盖了描述的动物物种1的70%以上。长期以来，由于其作为实验室模型的合适特征，如从胚胎到生殖成人的寿命相对较短（2.5-3.0个月），因此主要用于研究昆虫生理的祖先特征：图1A）和一个简单的繁殖。在过去的三十年里，它的使用已经扩大，以研究各种特征的祖先特征，如身体计划，神经分化，昼夜节律2，3，4。
先进的遗传工具在T.家政的应用可以进一步加速这种贡献，在广泛的研究领域。成功的RNA干扰（RNAi）介制的基因敲击在胚胎，仙女和成人已经报告在T.家庭4，5，6。系统性RNAi的效率仍然高度依赖物种-例如，它通常是高在科洛普泰拉，而它是低在麻风病顺序7。T. 家政的 RNAi 击倒的效率和持续时间尚未评估。除了RNAi，我们之前已经报告了成功的CRISPR/Cas9介质基因敲除在T.国内a8。CRISPR/Cas系统已广泛应用于昆虫的基因组编辑，特别是针对基因敲除。在建立CRISPR/Cas系统组件进入核9的协议后，通过敲击外源构造，可以扩大其用途，用于其他应用，如基因报告器检测、细胞谱系跟踪和转录活性操纵。结合已公布的基因组组装10，在T.家谱中广泛使用和进一步发展CRISPR/Cas为基础的基因组编辑将有助于研究昆虫卓越适应性成功背后的进化机制。在这里，我们描述了胚胎微射和交配成人T.家用，以产生突变株使用CRISPR/Cas9的详细协议。考虑到这种新方法，我们讨论了考虑非传统模型物种的独特生物学对于这些技术的成功应用的重要性。

<table>
	<tbody>
		<tr>
			<td>24-well plate</td>
			<td>Corning</td>
			<td>83-3738</td>
			<td></td>
		</tr>
		<tr>
			<td>Alt-R S.p. HiFi Cas9 Nuclease V3, 100 &#181;g</td>
			<td>Integrated DNA Technologies</td>
			<td>1081060</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-static cleaner</td>
			<td>Hozan</td>
			<td>Z-292</td>
			<td>for removing static electricity from a 24-well plate</td>
		</tr>
		<tr>
			<td>Barrier Box 20.7L</td>
			<td>AS ONE</td>
			<td>4-5606-01</td>
			<td>Large container</td>
		</tr>
		<tr>
			<td>FemtoJet 4i</td>
			<td>Eppendorf</td>
			<td>5252000013</td>
			<td>Electronic microinjector</td>
		</tr>
		<tr>
			<td>Femtotip II, injection capillary</td>
			<td>Eppendorf</td>
			<td>5242957000</td>
			<td>Glass injection capillary</td>
		</tr>
		<tr>
			<td>High Pack 2440mL</td>
			<td>AS ONE</td>
			<td>5-068-25</td>
			<td>Middle-sized container</td>
		</tr>
		<tr>
			<td>Incubator</td>
			<td>Panasonic</td>
			<td>MIR-554-PJ</td>
			<td>for 37 &#176;C incubation. No need to humidify inside the incubator.</td>
		</tr>
		<tr>
			<td>KOD Fx Neo</td>
			<td>Toyobo</td>
			<td>KFX-101</td>
			<td>PCR enzyme for genotyping. Optimized for an amplification from crude templates.</td>
		</tr>
		<tr>
			<td>Magnetic stand</td>
			<td>Narishige</td>
			<td>GJ-8</td>
			<td>for holding the micromanipulator</td>
		</tr>
		<tr>
			<td>Microloader</td>
			<td>Eppendorf</td>
			<td>5242956003</td>
			<td></td>
		</tr>
		<tr>
			<td>Micromanipulator</td>
			<td>Narishige</td>
			<td>MM-3</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope</td>
			<td>Olympus</td>
			<td>SZX12</td>
			<td>for microinjection. More than 35X magnification is sufficient for the microinjection</td>
		</tr>
		<tr>
			<td>MultiNA</td>
			<td>Shimadzu</td>
			<td>MCE-202</td>
			<td>Microchip electrophoresis system</td>
		</tr>
		<tr>
			<td>NiceTac</td>
			<td>Nichiban</td>
			<td>NW-5</td>
			<td>Double-sided tape to place eggs on a glass slide</td>
		</tr>
		<tr>
			<td>Paint brush (horse hair)</td>
			<td>Pentel</td>
			<td>ZBS1-0</td>
			<td></td>
		</tr>
		<tr>
			<td>Plant culture dish</td>
			<td>SPL Life Sciences</td>
			<td>310100</td>
			<td>Mating dish and water supplies for a large and middle-sized containers</td>
		</tr>
		<tr>
			<td>Proteinase K, recombinant, PCR Grade Lyophilizate from Pichia pastoris</td>
			<td>Roche</td>
			<td>3115836001</td>
			<td></td>
		</tr>
		<tr>
			<td>SZX 12 microscope</td>
			<td>Olympus</td>
			<td>SZX 12</td>
			<td>More than 35X magnification is sufficient for the microinjection</td>
		</tr>
		<tr>
			<td>Talcum powder</td>
			<td>Maruishi</td>
			<td>877113</td>
		</tr>
		<tr>
			<td>Tetra Goldfish Gold Growth</td>
			<td>Spectrum Brands</td>
			<td></td>
			<td>Artificial regular fish food</td>
		</tr>
	</tbody>
</table>

egg microinjection and efficient mating for genome editing in the firebrat thermobia domestica

火布拉特 热比亚家是 一种无翼的无翼物种，适合研究昆虫的发展机制，导致它们在地球上成功的进化辐射。基因工具（如基因组编辑）的应用是理解基因变化的关键，这些变化是 Evo-Devo 方法中负责进化过渡的关键。在这篇文章中，我们描述了我们目前的协议，以产生和维持 T.国内突变菌株。我们报告一种干注射方法，作为报告的湿注射方法的替代品，使我们能够获得注射胚胎的稳定高存活率。我们还报告了一个优化的环境设置，以配合成人，并获得后代的高效率。我们的方法强调了将每个物种独特的生物学考虑在非传统模型生物中成功应用基因组编辑方法的重要性。我们预测，这些基因组编辑协议将有助于实施 T.家用作为 实验室模型，并进一步加快该物种中有用的遗传工具的开发和应用。

在我们手中，大约100个鸡蛋可以很好地注射一个单一的注射毛细亚时，它有足够的尖端（图3C）。在产卵后的前8小时内在胚胎中注射gRNA/Cas9核糖核蛋白复合物，导致在gRNA靶点产生因德尔。这会导致注射一代 （G0） 的某些细胞发生胆汁突变，因此在 G0 中通常获得突变马赛克表型。例如，当本协议用于注射旨在瞄准 白色 基因的 gRNA 时，32.6% 的 G0 仙女在其复合眼睛和背?...

Watch this Scientific Journal Video about Egg Microinjection and Efficient Mating for Genome Editing in the Firebrat Thermobia domestica at JoVE.com

Egg Microinjection and Efficient Mating for Genome Editing in the Firebrat Thermobia domestica

我们为卵子的饲养、微喷射和在基因组编辑后生成和维持突变菌株的火布拉特 热比亚家用菌 株的有效交配提供了详细的协议。

卵子微弹射和有效交配的基因组编辑在火布拉特 热比亚家

The firebrat Thermobia domestica is an ametabolous, wingless species that is suitable for studying the developmental mechanisms of insects that led to ...

Research

JoVE Journal

Developmental Biology

Egg Microinjection and Efficient Mating for Genome Editing in the Firebrat Thermobia domestica

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Efficient Gene Transfer in Chick Retinas for Primary Cell Culture Studies: An Ex-ovo Electroporation Approach

在小鸡视网膜有效的基因转移的原代细胞培养研究：<em&gt;前OVO</em&gt;电穿孔法

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The zebrafish model is an emerging system for the study of neuromuscular disorders.  In the study of neuromuscular diseases, the integrity of the muscle ...

Technique to Target Microinjection to the Developing Xenopus Kidney

Technique to Target Microinjection to the Developing Xenopus Kidney

技术目标显微注射到显影<em&gt;爪</em&gt;肾

The embryonic kidney of Xenopus&#160;laevis (frog), the pronephros, consists of a single nephron, and can be used as a model for kidney disease. Xenopus ...

Identification of Critical Conditions for Immunostaining in the Pea Aphid Embryos: Increasing Tissue Permeability and Decreasing Background Staining

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The pea aphid Acyrthosiphon pisum, with a sequenced genome and abundant phenotypic plasticity, has become an emerging model for genomic and developmental ...

Microinjection for Transgenesis and Genome Editing in Threespine Sticklebacks

显微注射转基因的基因组，并在编辑Threespine刺鱼

The threespine stickleback fish has emerged as a powerful system to study the genetic basis of a wide variety of morphological, physiological, and ...

Embryo Microinjection and Electroporation in the Chordate Ciona intestinalis

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胚胎显微注射和电的脊索动物<em&gt;玻璃海鞘</em

Simple model organisms are instrumental for in vivo studies of developmental and cellular differentiation processes. Currently, the evolutionary distance ...

Collection of Post-mating Semen from the Female Reproductive Tract and Measurement of Semen Liquefaction in Mice

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In mice, ejaculated semen is deposited in the uterus. After ejaculation, the semen changes consistency from gel-like to watery, a process called ...

An Efficient Strategy for Generating Tissue-specific Binary Transcription Systems in Drosophila by Genome Editing

An Efficient Strategy for Generating Tissue-specific Binary Transcription Systems in Drosophila by Genome Editing

基于基因组编辑的果蝇组织特异二进制转录系统的高效生成策略

Binary transcription systems are powerful genetic tools widely used for visualizing and manipulating cell fate and gene expression in specific groups of ...

Microinjection-based System for In Vivo Implantation of Embryonic Cardiomyocytes in the Avian Embryo

基于微注射的免疫胚胎心肌细胞体内植入系统

Interpreting the relative impact of cell autonomous patterning versus extrinsic microenvironmental influence on cell lineage determination represents a ...

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Life is spatially partitioned within lipid membranes to allow the isolated formation of distinct molecular states inside cells and organelles. Cell fusion ...

卵子微弹射和有效交配的基因组编辑在火布拉特 热比亚家

卵子微弹射和有效交配的基因组编辑在火布拉特热比亚家