Name: generation of chimeric axolotls with mutant haploid limbs through embryonic grafting
Uploaded: 2020-01-29T13:00:00
Description: Watch this Scientific Journal Video about Generation of Chimeric Axolotls with Mutant Haploid Limbs Through Embryonic Grafting at JoVE.com

我们要感谢凯瑟琳·罗伯茨对阿索洛特尔殖民地的关心。这项工作的资金由康涅狄格州创新再生医学研究基金（15RMA-YALE-09和15元人民币-YALE-01）和尤尼斯·肯尼迪·施莱佛国家儿童健康和人类发展研究所（个人博士后）提供。奖学金 F32HD086942）。

该协议中使用的实验程序由耶鲁大学机构动物护理和使用委员会（IACUC，2017–10557）批准，并符合所有联邦关于脊椎动物使用的政策和准则。所有动物实验都是在耶鲁大学的设施中对墨西哥的阿姆比托马（axolotls）进行的。
1. 双倍体胚胎生成
<ol><li>获得GFP®二倍体胚胎，作为移植宿主通过自然交配使用一个或两个gfp父母24。</li>
	<li>收集新鲜铺?...

<ol>
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Stem Cells International. 2017, 1-6 (2017).</li><li>Khattak, S., 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=Optimized+axolotl+(Ambystoma+mexicanum)+husbandry,+breeding,+metamorphosis,+transgenesis+and+tamoxifen-mediated+recombination.">Optimized axolotl (Ambystoma mexicanum) husbandry, breeding, metamorphosis, transgenesis and tamoxifen-mediated recombination.</a> Nature Protocols. 9 (3), 529-540 (2014).</li><li>Vachon, P., Zullian, C., Dodelet-Devillers, A., Roy, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+the+anesthetic+effects+of+MS222+in+the+adult+Mexican+axolotl+(Ambystoma+mexicanum).">Evaluation of the anesthetic effects of MS222 in the adult Mexican axolotl (Ambystoma mexicanum).</a> Veterinary Medicine: Research and Reports. 7, 1-7 (2016).</li><li>Montague, T. 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在我们的协议中，有几个关键步骤用于生成单倍体-双倍体嵌合体，操作技术人员应考虑这些步骤，以便获得一致的嫁接结果。
单倍体生成失败的最可能原因是体外激活条件不佳。必须使用适当数量的活化精子来激活卵子。为了延长活力，精子样本应始终保持在4°C。在将任何精子样本涂在卵子上之前，使用倒置显微镜检查精子的存活性。完全非活动性精子绝不应使用，浓度应...

从历史上看，两栖动物的胚胎组织移植一直是探索发育生物学和再生的基本机制的重要技术。axolotl是一种蜥蜴，在受伤或截肢后，具有再生组织和复杂结构（如四肢和器官）的惊人能力。同样令人印象深刻的是，他们可以在胚胎期、幼年期和成人期1、2、3接受其他个体的组织移植，而不会被拒绝。产生整个结构的胚胎区域，如四肢、尾巴、眼睛和头部，以及更特定的组织，如神经切除和索米特，可以在胚胎之间移植，以产生嵌合动物1，2，4，5，6。近一个世纪以来，对这种嵌合动物的研究为再生、组织分化、尺寸控制和模式1、7、8提供了重要的见解。
在过去的十年里，许多再生组织的转录研究已经产生了对基础蜥蜴再生9，10，11，12，13的遗传程序的见解。这些研究增加了候选基因的扩大列表，迄今为止，这些基因在再生背景下基本上没有特征。靶向诱变技术，如CRISPR/Cas，现在允许研究这种基因，这种基因方法大大促进了大轴醇基因组14，15，16的最近测序和组装。
我们寻求开发将经典发育生物学与新的基因技术相结合的技术，以解剖再生机制。产生单倍体胚胎的方法已经建立了几十年。虽然这些技术一直被认为是作为遗传模型生物18的蜥蜴的优势，但随后的遗传研究很少纳入单倍体动物。我们使用体外活化在axolotl生产单倍体胚胎，作为组织捐赠者移植19。利用携带荧光遗传标记的胚胎，我们设计了可靠的方法来生成几乎完全来自供体组织的四肢（图1A）。通过结合这两种技术，我们绕过了与单倍体相关的晚期胚胎杀伤力，允许生产完全发育的、移植的单倍体四肢（图1B，图1B'和图2）。
通过在移植前在单倍体胚胎中进行CRISPR/Cas介导的诱变，以产生具有突变单倍体四肢的嵌合体轴片，我们可以在肢体发育和再生的背景下专门研究基因功能。这允许从潜在的胚胎致命变异表型中拯救四肢。虽然CRISPR/Cas显微注射可以产生高度突变的动物，这类动物通常是高度马赛克，在目标部位14、20上保留一定程度的野生型等位基因和各种不同的突变。单倍体细胞中基于CRISPR的诱变增加单等位基因丧失功能突变的渗透，因为它们不能被保留的野生型等位基因掩盖。因此，在单倍体细胞系中基于CRISPR的筛选越来越多地用于研究许多细胞过程21、22、23的遗传基础。通过将基于CRISPR的谱系追踪与我们的单倍体肢体芽移植方案相结合，本文描述的方法可以作为活动物中单倍体遗传屏幕的平台。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>#55 Dumont Forceps</td>
			<td>Fine Science Tools</td>
			<td>11295-1</td>
			<td>Only use Dumostar material (can be autoclaved)</td>
		</tr>
		<tr>
			<td>Amphotericin B</td>
			<td>Sigma Aldrich</td>
			<td>A2942-20ML</td>
			<td>20 mL</td>
		</tr>
		<tr>
			<td>Antibiotic-Antimycotic 100x</td>
			<td>Thermo Fisher</td>
			<td>15240062</td>
			<td></td>
		</tr>
		<tr>
			<td>Ciprofloxacin</td>
			<td>Sigma Aldrich</td>
			<td>17850-5G-F</td>
			<td></td>
		</tr>
		<tr>
			<td>Ficoll 400 (polysucrose 400)</td>
			<td>bioworld</td>
			<td>40600032-3</td>
			<td>Ficoll 400</td>
		</tr>
		<tr>
			<td>Gentamicin</td>
			<td>Sigma Aldrich</td>
			<td>G1914-250MG</td>
			<td></td>
		</tr>
		<tr>
			<td>Heating/Cooling Incubator</td>
			<td>RevSci</td>
			<td>RS-IF-233</td>
			<td></td>
		</tr>
		<tr>
			<td>Human Chorionic Gonadotropin</td>
			<td>Merk</td>
			<td></td>
			<td>Chorulon</td>
		</tr>
		<tr>
			<td>Megascript T7 Transcription Kit</td>
			<td>Thermo Fisher</td>
			<td>AM1334</td>
			<td>40 reactions</td>
		</tr>
		<tr>
			<td>Miroscope Cooling Stage</td>
			<td>Brook Industries</td>
			<td>Custom</td>
			<td>Custom</td>
		</tr>
		<tr>
			<td>NLS Cas9 Protein</td>
			<td>PNAbio</td>
			<td>CP01-200</td>
			<td>4 vials of 50 &#181;g protein each</td>
		</tr>
		<tr>
			<td>Plasmocin</td>
			<td>Invivogen</td>
			<td>ant-mpt-1</td>
			<td>Treatment level</td>
		</tr>
		<tr>
			<td>Recipes</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>1.0x Marc&#39;s modified Ringer&#39;s solution (MMR)</td>
			<td></td>
			<td></td>
			<td>0.1 M NaCl, 2 mM KCl, 1 mM MgSO4, 2 mM CaCl2, 0.1 mM EDTA, 5 mM HEPES (pH 7.8), ph 7.4</td>
		</tr>
		<tr>
			<td>40% Holtfreter&#39;s solution</td>
			<td></td>
			<td></td>
			<td>20 mM NaCl, 0.2 mM KCl, 0.8 mM NaHCO3, 0.2 mM CaCl2, 4 mM MgSO4, pH to 7.4</td>
		</tr>
	</tbody>
</table>

generation of chimeric axolotls with mutant haploid limbs through embryonic grafting

越来越多的基因技术和资源使研究人员能够探索某些种类的蜥蜴（如阿索洛特人）在成年后再生整个四肢的能力的分子起源。在这里，我们概述了用于生成具有Cas9诱变性单倍体前肢的嵌合轴球的技术，可用于探索基因功能和肢体再生的保真度。我们将多种胚胎学和遗传技术相结合，包括通过体外活化、CRISPR/Cas9诱变生成和组织移植到一个协议中，在再生模型生物体中产生单倍体遗传筛选的独特系统。这种策略减少了肢体再生中基因功能分析所需的动物数量、空间和时间。这也允许研究其他基本过程（如器官形成、组织形态生成和其他基本胚胎过程）可能需要的基因的再生特定功能。本文描述的方法是在脊椎动物模型系统中进行单倍体遗传筛选的独特平台。

开发单倍体胚胎可以通过其"单倍体综合征"表型29来区分双倍体胚胎。在移植阶段，单倍体胚胎沿前后轴和蛋黄塞的不完全外壳表现出较低的曲率（图3A）。荧光显微镜可用于确保单倍体胚胎不含父系衍生的GFP表达（图3B）。
当移植物清洁?...

Watch this Scientific Journal Video about Generation of Chimeric Axolotls with Mutant Haploid Limbs Through Embryonic Grafting at JoVE.com

Generation of Chimeric Axolotls with Mutant Haploid Limbs Through Embryonic Grafting

该协议的目标是使用胚胎组织移植技术，从Cas9诱变的供体组织中提取单倍体前肢的嵌合体轴片。

通过胚胎移植，生成具有突变倍体肢体的奇美体轴球

A growing set of genetic techniques and resources enable researchers to probe the molecular origins of the ability of some species of salamanders, such as ...

该协议允许生产可用于轴球基因筛选的变异单倍体四肢。在单倍体中，即使是低层次的突变，也会产生细胞内功能表型的丧失。这种嫁接技术也可以用来拯救胚胎致命的表型。这种方法可用于研究任何两栖动物的再生，这些两栖物种都可用于体外受精和嫁接。在使用单倍体胚胎作为捐赠者之前，我们建议练习并确认GP阳性和负二倍体胚胎之间的移植成功。对于女性游戏捐赠者的准备，麻醉性成熟的白色或白色RFP女性斧头作为游戏捐赠者。在确认对疼痛模拟缺乏反应后，使用30量胰岛素注射器将0.15毫升的人类胆道性腺激素注射到后肢的肌肉背。将注射器以 45 度角插入中线，以避免与脊髓接触。将注射的动物放在新鲜的40%霍尔特弗雷特溶液中，将斧头转移到8至12摄氏度的冰箱中48小时。然后将雌性返回到室温。当雌性正在产卵时，将一个完全麻醉的雄性放在湿纸巾的上部位置，放在解剖显微镜下，用占统治地位的手将P1000移液器的尖端放在基卡底部。将另一个食指和拇指放在骨盆两到三厘米的罗体，轻轻挤压动物，同时将手指向后腿移动，将收集每个样本的精子尿样冲洗到新的微离心管中。然后将每个样品中的五微升转移到培养皿中，使用倒置显微镜检查精子的质量。计数后，在0.1X无菌MMR中将精子稀释至每毫升8倍至第四细胞的浓度，并每卵将0.5微升精子细胞转移到新的培养皿中。然后使用移液器尖端将悬浮液铺到一毫米厚的层，并使用塑料升降机将样品从 254 纳米交联器的灯泡中放置四厘米，以每毫米平方的 8 倍 10 到 5 微焦耳进行辐照。收集健康的精子样本后，将雌性放在一叠湿纸巾上的苏平位置，并使用类似的冲洗运动从雌性斧头中提取未受精的卵子。然后用湿钳将鸡蛋转移到10厘米的培养皿中。在收集后15分钟内，使用P10移液器将辐照精子分配到未受精的卵子上，每个卵子上涂有0.25至0.5微升的灭活精子。让鸡蛋在室温下坐30分钟后，用0.1XMMR将鸡蛋淹没。水化后30分钟，用锋利的钳子对鸡蛋进行解热，并在7小时内将鸡蛋放在18摄氏度的微注射中。要产生单倍体双倍体嵌合，将一个健康单倍体捐赠者与一个或两个阶段匹配的GP正双倍体宿主胚胎放在含有手术介质的预冷手术盘中的槽中，在10摄氏度或较低的冷却阶段。使用两个超细的高压直头钳子去除整个肢体芽和周围的外皮和中皮组织层，并留出宿主组织。从单倍体捐赠者身上取出同等大小的组织护套，将单倍体供体组织护套放在宿主胚胎的相应区域。用从碎显微镜盖玻璃上用自动处理的矩形玻璃碎片固定组织，然后轻轻地将组织压入宿主胚胎体。将锚留在位，每 20 分钟检查 60 到 75 分钟，以确认玻璃在使用钳子剥去盖玻璃碎片之前没有滑动。将移植的胚胎移植到新鲜的手术介质中，使其在8至12摄氏度下一夜之间痊愈，然后将植入的胚胎单独放在12或24孔的板中。38.7%的卵母细胞发育成正常发育的单倍体胚胎。在移植阶段，单倍体胚胎沿前后轴显示曲线降低，蛋黄塞的外壳不完整。此外，荧光显微镜可用于确认单倍体胚胎没有亲子衍生的GP表达。失败和不纯的嫁接产生各种表型。当移植物是清洁和正常发展，GFP表达应主要限于胸膜丛，神经网络派生自主机脊髓。Punctate GFP表达也存在于单个细胞中，这些细胞似乎是血液衍生宿主细胞中迁移到发育中的肢体的感官神经元。当使用 RFP 阳性捐赠者时，单倍体移植四肢表现出 RFP 的通用表达。在整个发育过程中，非突变的单倍体四肢明显短于嵌合动物中相对的二倍体前肢。非突变的单倍体四肢完全再生，尽管与二倍体相比，它们在达到数字生长阶段方面略有延迟。记得使用无菌技术，完全去除和更换胚胎宿主中皮和外皮组织层，以产生一个干净的移植肢体。突变的四肢可以截肢，并评分再生表型。突变等位基因可以用下一代测序进行量化。结合CRISPR-Cas9突变，我们的移植技术使我们能够在再生单倍体四肢时对靶向基因进行负选择筛选。

Research

JoVE Journal

Genetics

The Terroir Concept Interpreted through Grape Berry Metabolomics and Transcriptomics

风土通过概念葡萄果实代谢组学和转录组解释的

Terroir refers to the combination of environmental factors that affect the characteristics of crops such as grapevine (Vitis vinifera) according to ...

科研

遗传学

Generation of Fluorescent Protein Fusions in Candida Species

Generation of Fluorescent Protein Fusions in Candida Species

在荧光蛋白融合的一代<em&gt;念珠菌</em&gt;物种

Candida species, prevalent colonizers of the intestinal and genitourinary tracts, are the cause of the majority of invasive fungal infections in humans. ...

Controllable Ion Channel Expression through Inducible Transient Transfection

通过诱导瞬时转染可控离子通道表达

Transfection, the delivery of foreign nucleic acids into a cell, is a powerful tool in protein research. Through this method, ion channels can be ...

Generation of Native Chromatin Immunoprecipitation Sequencing Libraries for Nucleosome Density Analysis

核密度分析的天然染色质沉淀测序文库的生成

We present a modified native chromatin immunoprecipitation sequencing (ChIP-seq) experimental protocol compatible with a Gaussian mixture distribution ...

Chromatin Immunoprecipitation (ChIP) Protocol for Low-abundance Embryonic Samples

Chromatin Immunoprecipitation ChIP Protocol for Low-abundance Embryonic Samples

低丰度胚胎标本的染色质沉淀 (芯片) 协议

Chromatin immunoprecipitation (ChIP) is a widely-used technique for mapping the localization of post-translationally modified histones, histone variants, ...

Generation of a Gene-disrupted Streptococcus mutans Strain Without Gene Cloning

Generation of a Gene-disrupted Streptococcus mutans Strain Without Gene Cloning

无基因克隆的基因破坏的变形链球菌菌株的生成

Typical methods for the elucidation of the function of a particular gene involve comparative phenotypic analyses of the wild-type strain and a strain in ...

Amplification of Near Full-length HIV-1 Proviruses for Next-Generation Sequencing

用于下一代测序的近全长 HIV-1 Proviruses 的扩增

The Full-Length Individual Proviral Sequencing (FLIPS) assay is an efficient and high-throughput method designed to amplify and sequence single, near ...

Generation of Tumor Organoids from Genetically Engineered Mouse Models of Prostate Cancer

从前列腺癌的遗传工程小鼠模型生成肿瘤有机体

Methods based on homologous recombination to modify genes have significantly furthered biological research. Genetically engineered mouse models (GEMMs) ...

Using Next Generation Sequencing to Identify Mutations Associated with Repair of a CAS9-induced Double Strand Break Near the CD4 Promoter

使用下一代测序来鉴定与CD4启动子附近CAS9诱导的双链断裂修复相关的突变

Double strand breaks (DSBs) in DNA are the most cytotoxic type of DNA damage. Because a myriad of insults can result in these lesions (e.g., replication ...