Name: induction and evaluation of inbreeding crosses using the ant, vollenhovia emeryi
Uploaded: 2018-10-05T15:00:00
Description: Watch this Scientific Journal Video about Induction and Evaluation of Inbreeding Crosses Using the Ant, Vollenhovia Emeryi at JoVE.com

我们感谢日本东京 AntRoom 的代表 Taku 岛先生向我们提供了他的五 emeryi reproductives 的照片。该项目由日本促进科学学会 (16J00011) 为青年科学家研究金和资助青年科学家 (B) (16K18626) 提供资金。

1. 在实验室中Emeryi殖民地的实地收集和维持
注: 在日本各地, 腐烂的原木和腐烂的树木 branchesin 次生森林中发现了v emeryi的巢。这一物种显示了两种类型的殖民地,即(1) 只生产长翅皇后和 (2) 殖民地主要生产短翅皇后除了小数目长翅皇后8,14。在本议定书中, 我们收集了日本石川县的后一类殖民地。
<ol><li>在初夏收...

<ol>
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本文论证了可用于诱导近亲繁殖杂交的协议, 并对emeryi中近亲繁殖的发生进行了评价。在实验中, 用于交叉的个体的基因分型是确保近亲繁殖杂交成功的必要条件。然而, 这些交叉试验的有效性显然是显而易见的, 因为双雄动物全年都可以生产, 而单倍体雄性只能在秋季在田间和实验室6生产。同胞交配皇后开始在交叉后立即产生雄性后代。在v. emeryi6<...

群居 Hymenopteran 类群, 如蚂蚁和蜜蜂, 进化出了一个 haplodiploid 的性别决定体系, 在这种系统中, 异型在一个或多个补充性决心 (CSD) 的个体成为女性, 而那些是同性恋者或 hemizygous成为男性 (图 1A)1。
基因和分子成分参与性别决定级联已被很好的研究在蜜蜂, api 蜜蜂, hymenopteran 模型生物2,3,4。最近的比较基因组调查显示, 蚂蚁和蜜蜂在性别决定途径中分享了许多假定的同系物, 如最初的性别决定基因, csd5。然而, 这些同系物的功能保护的证据仍然缺乏蚂蚁。
为了解决这一问题, 必须发展近亲繁殖线, 因为它们对遗传图谱和分子研究是必不可少的。然而 , 由于生命周期的复杂性质已经演变 , 很难在实验室中维持和进行实验叉。
在这里, 我们使用Vollenhovia emeryi作为一个模型, 以调查的遗传和分子基础的性别决定系统的蚂蚁6,7。本种的近亲繁殖系是在蚂蚁6中首次为与性别决定有关的性状的数量性状基因位点 (QTL) 的连锁映射而开发的。此外, 还研究了7的分子性别测定级联。这个物种进化出了一种不寻常的繁殖系统, 既使用了孤雌生殖和雄核发育 (图 1B)8,9。大多数新的皇后和男性是无性生产的母体和父系基因组, 分别。此外, 工人和一些皇后生产性8。这种生殖系统特别适合于遗传学研究, 因为近亲交配产生的杂交后代使用性生产的皇后和雄性的基因等同于传统的回交。由于性生产的皇后不同形态上的皇后从母体基因组10 (图 1B), 进行和评估近亲繁殖交叉是大大简化了使用这种方法。
本文建立了交叉试验实验菌落的确定方法, 利用全同胞对对近亲繁殖交叉口的应用, 并利用群体成员的基因分型和男性后代的解剖评价这些杂交的成功与否生殖器在 v. emeryi中被描述。
无论采用何种生殖系统, 近交交叉的应用往往是任何研究膜翅目性测定系统的必要的第一步。例如, 在Cardiocondyla obscurior, 在实验室里, 10 代全同胞交配后几乎完全没有双雄雄性, 这表明了惩教署所在地11的缺席。有可能预测惩教署基因座的数目, 从近亲交配产生的男性比例6,12,13。

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			<td style="width:223px;">Any brand can be used</td>
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			<td height="45" style="height:45px;width:392px;">Dry Cricket diet</td>
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			<td height="45" style="height:45px;width:392px;">Brown shuger&#160;</td>
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			<td>Any brand can be used</td>
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			<td height="45" style="height:45px;width:392px;">Styrene Square-Shaped Case</td>
			<td style="width:239px;">AS ONE</td>
			<td style="width:185px;">Any size</td>
			<td>Size varies by number of ants</td>
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			<td height="45" style="height:45px;width:392px;">Incbator</td>
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			<td height="45" style="height:45px;width:392px;">1.5mL Hyper Microtube,Clear, Round bottom</td>
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			<td height="45" style="height:45px;width:392px;">-Cellstain- DAPI solution</td>
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			<td height="64" style="height:64px;width:392px;">VECTASHIELD Hard&#12539;Set Mounting Medium with TRITC-Phalloidin</td>
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			<td style="width:185px;">H-1600</td>
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induction and evaluation of inbreeding crosses using the ant, vollenhovia emeryi

在 hymenopteran 模型生物蜜蜂中, 在蜜蜂中广泛研究了性别决定级联的遗传和分子成分。然而, 对于其他非模型 hymenopteran 类群中发现的性别决定机制, 如蚂蚁, 知之甚少。由于 hymenopteran 物种进化的生命周期的复杂性质, 在实验室中很难维持和进行实验性的交叉。在这里, 我们描述了进行近亲繁殖杂交的方法和评估这些杂交在蚂蚁Vollenhovia emeryi的成功。利用v. emeryi在实验室诱导近亲繁殖, 相对简单, 因为该物种具有独特的生物学特性。具体地说, 这个物种产生雄性雄性, 而雌性 reproductives 表现出翅膀多态性, 简化了遗传杂交中表型的识别。此外, 评估近亲繁殖的成功是直接的, 因为雄性可以连续生产的近亲繁殖十字架, 而正常的雄性只出现在一个明确定义的生殖季节在田间。我们的协议允许以v. emeryi为模型, 研究蚂蚁物种性别决定系统的遗传和分子基础。

利用 f0和 f1代的微卫星分析结果表明, 近交杂交成功产生 (图 4)6。由于近亲繁殖十字架 , 交配皇后在建立实验叉殖民地的一个月之内获得。一个处所 (27.1 8.91% SD) 所有子代 (F2) 从近亲繁殖十字架是男性, 而剩下的人是女性 (工作者和女王)6。利用近亲繁殖杂交后代的 QTL 图谱表明, 近交杂交产?...

Watch this Scientific Journal Video about Induction and Evaluation of Inbreeding Crosses Using the Ant, Vollenhovia Emeryi at JoVE.com

Induction and Evaluation of Inbreeding Crosses Using the Ant, Vollenhovia Emeryi

在本议定书中, 介绍了进行近亲繁殖杂交的方法, 并对这些杂交的成功进行了描述, 为蚂蚁Vollenhovia emeryi。这些协议对于旨在了解膜翅目中性别决定系统的遗传基础的实验是很重要的。

利用蚂蚁Vollenhovia Emeryi对近亲繁殖杂交的诱导和评价

The genetic and molecular components of the sex-determination cascade have been extensively studied in the honeybee, Apis mellifera, a hymenopteran model ...

这种方法可以帮助回答分子生物学和海月经的遗传领域，如性测定系统的关键问题。这项技术的主要优点是，我们可以轻松地诱导近亲繁殖线对蚂蚁Vollenhovia Emery，这是必要的基因映射和分子状态。因此，这个任务可以提供特定蚂蚁的性别确定系统的见解，它也可以适用于其他蚂蚁和其他海月视物种，如黄蜂。要开始该协议，收集V.Emeryi殖民地在初夏。使用吸气器将蚂蚁标本从采集的树枝转移到带玻璃盖的人工石膏巢中。在16至8小时的光暗周期内，将人工巢中的菌落保持在25摄氏度。提供自来水与洗涤瓶，以保持石膏湿润每隔一天在同一时间。接下来，添加约100微克用铝箔包裹的干板球粉和红糖水充满尖端到殖民地，每隔一天使用20微升尖，直到新的生殖蚂蚁出现。重要的是要收集微殖民地从田野和为他们提供足够的食物，以获得新的女王和男性。首先，通过将殖民地放置在控制环境设定在摄氏4度的房间里15分钟，阻止个人移动。在立体镜下用钳子去除 30 只工人蚂蚁的中腿，以区分 F-Zero 代代和后续近亲繁殖十字架中产生的工人。然后将蚂蚁转移到一个新的较小的石膏巢近亲繁殖十字架。接下来，在含有工人的石膏巢中加入三到四个幼虫或阴性。将一个长翼女王和一只雄性转移到先前准备的石膏巢中， 用于近亲繁殖的十字架。对于这一步骤，重要的是保持实验交叉殖民地在同一状态，作为一个正常的殖民地。仅仅通过将男性和女性放在一起来繁殖是很难诱导的。在16到8小时的光光下，将殖民地保持25摄氏度，黑暗循环，食物和水供应，直到女王失去翅膀产卵。每天在立体显微镜下检查实验殖民地。在F-One后代之间设置近亲繁殖十字架后，可以在立体镜下观察卵子。在F-One女王开始产卵后，从巢中去除F-one雄性和幼虫或阴性，以防止F-一代和F-2一代混合。保持殖民地在相同的条件下，直到F-2后代出现。用钳子拆下 F-零女王的一条腿。将腿部转移到含有100微升冷却剂的1.5毫升微管中。在立体镜下，用钳子解剖一个装满300微升超纯水的玻璃盘中的女性腹部。与含有雌性男性精子的精子不同。剥去精子的组织，用昆虫针将精子从女性组织中分离。使用微饼垫，将精子转移到含有100微升的冷却剂的1.5毫升微管中。在95摄氏度下孵育F-Zero女王和精子的样本20分钟。闪存中点熔断微管，将样品储存在四摄氏度。在确认卵子生产后，由siv-matef-One女王，提取女王的DNA使用棚翅膀或一个中腿和基因型。接下来，通过一条腿的基因分型提取F-1男性的DNA。为了评估近亲繁殖十字架中雄性蚂蚁的生育能力，用钳子用400微升PBS溶液在玻璃盘中解剖内部生殖器官。然后取出 PBS，并使用微饼垫将其替换为 4% 的甲醛。通过将样品在PFA中孵育40分钟，在室温下固定组织。固定后，使用微饼垫用400微升PBS洗涤组织五次。将 DAPI 溶液稀释至 PBS 中每毫升 1 毫克。去除 PBS，向组织添加约 300 微升稀释的 DAPI 染色溶液。在室温的黑暗条件下孵育组织15分钟。孵育后，用400微升PBS洗涤组织五次，用钳子将组织转移到玻璃滑梯的中心。然后将组织安装在含有 TRITC 四甲苯胺的安装介质上。最后使用20或60 3倍物镜用共声激光扫描显微镜观察样品。Haploid 雄性 v Emeryi 睾丸的微观图像显示健康的纤维组织或精子，在双倍男性 v Emeryi 中不存在，没有显示健康的纤维组织。双倍体雄性v Emeryi的男性生殖器官没有产生精子，他们的睾丸也没有发育，这表明在近亲繁殖十字架中产生的雄性生殖器官是无菌的。在它的发展，这项技术铺平了道路，我们探索性米阿托尼在Vollenhovia埃默里伊的快速定时蚂蚁物种的恒定。

Research

JoVE Journal

Genetics

Induction and Evaluation of Inbreeding Crosses Using the Ant, Vollenhovia Emeryi

Procedure for Adaptive Laboratory Evolution of Microorganisms Using a Chemostat

程序的微生物实验室自适应演化的恒化

Natural evolution involves genetic diversity such as environmental change and a selection between small populations. Adaptive laboratory evolution (ALE) ...

科研

遗传学

Detection of Copy Number Alterations Using Single Cell Sequencing

单细胞测序拷贝数改变的检测

Detection of genomic changes at single cell resolution is important for characterizing genetic heterogeneity and evolution in normal tissues, cancers, and ...

Analysis of the c-KIT Ligand Promoter Using Chromatin Immunoprecipitation

使用染色质免疫沉淀分析c-KIT配体启动子

Multiple cellular processes, including DNA replication and repair, DNA recombination, and gene expression, require interactions between proteins and DNA. ...

High Resolution Fluorescent In Situ Hybridization in Drosophila Embryos and Tissues Using Tyramide Signal Amplification

High Resolution Fluorescent In Situ Hybridization in Drosophila Embryos and Tissues Using Tyramide Signal Amplification

高分辨率荧光原位杂交在果蝇胚胎和组织中使用 Tyramide 信号放大

In our efforts to determine the patterns of expression and subcellular localization of Drosophila RNAs on a genome-wide basis, and in a variety of ...

Using In Vitro and In-cell SHAPE to Investigate Small Molecule Induced Pre-mRNA Structural Changes

利用体外和细胞内形状研究小分子诱导的预 mrna 结构变化

In the process of drug development of RNA-targeting small molecules, elucidating the structural changes upon their interactions with target RNA sequences ...

Evaluation of a Universal Nested Reverse Transcription Polymerase Chain Reaction for the Detection of Lyssaviruses

一种通用嵌套逆转录酶链反应检测裂解病毒的评价

To detect rabies virus and other member species of the genus Lyssavirus within the family Rhabdoviridae, the pan-lyssavirus nested reverse transcription ...

Use of Frozen Tissue in the Comet Assay for the Evaluation of DNA Damage

在彗星分析中使用冷冻组织评估DNA损伤

The comet assay is gaining popularity as a means to assess DNA damage in cultured cells and tissues, particularly following exposure to chemicals or other ...

Introducing a Gene Knockout Directly Into the Amastigote Stage of Trypanosoma cruzi Using the CRISPR/Cas9 System

Introducing a Gene Knockout Directly Into the Amastigote Stage of Trypanosoma cruzi Using the CRISPR/Cas9 System

使用CRISPR/Cas9系统将基因挖空直接引入锥虫瘤的肌瘤阶段

Trypanosoma cruzi is a pathogenic protozoan parasite that causes Chagas&#8217; disease mainly in Latin America. In order to identify a novel drug target ...

Generation of Defined Genomic Modifications Using CRISPR-CAS9 in Human Pluripotent Stem Cells

在人类多能干细胞中使用CRISPR-CAS9生成定义的基因组修饰

Human pluripotent stem cells offer a powerful system to study gene function and model specific mutations relevant to disease. The generation of precise ...

Embryo Injections for CRISPR-Mediated Mutagenesis in the Ant Harpegnathos saltator

Embryo Injections for CRISPR-Mediated Mutagenesis in the Ant Harpegnathos saltator

胚胎注射用于CRISPR介导的诱变在蚂蚁Harpegnathos盐化剂中

The unique traits of eusocial insects, such as social behavior and reproductive division of labor, are controlled by their genetic system. To address how ...