我们要感谢恰克的指导, 约翰·多德帮助制定这一育种战略, 马西埃沃克为她的工作, 完善的骨骼染色, Charline 沃克和邦尼乌尔曼为有益的斑马鱼的建议。这项工作得到了 K99/R00 DE024190 对 JTN 的支持。

本协议中描述的所有实验都是按照科罗拉多大学和俄勒冈大学机构动物保育和使用委员会 (IACUC) 的要求完成的. 
 1. 准备未选定的起始库存 <ol> <li> 通过鳍片剪辑 11 , 通过选择的方法对 full-sibling 动物的基因型基因进行分型, 以确定杂携带者的兴趣KASP 12 , 13 , 14 。此协议是使用斑马鱼 mef2ca b1086 应变执行的. </li>
	<li> KASP 基因分型 " 
	注: KASP 的等位基因特异性底漆是由提供试剂的公司设计的 (请参见 材料表 )。要获得适当的 6-羧基-X-罗丹明 (火箭) 染料浓度为特定的 real-time PCR 机访问公司网站。
	<ol> <li> 将斑马鱼的尾部组织放置在50和 #181; 溶解缓冲液 (10 毫米三 (pH 8.3), 50 毫米氯化钾, 1.5 毫米氯化镁 2 , 0.3% 非离子表面活性剂, 0.3% 苯基-聚乙二醇)。添加10和 #181; 10 毫克/毫升蛋白酶 K 每96井 PCR 板和消化在热循环在55和 #176; c 为 2-5 h, 后跟 20 min 94 和 #176; c 失活步骤。消化后将裂解产物冷藏. #160; 注: 裂解产品可在制备后几个月内成功使用。用50毫米氢氧化钠裂解组织时, KASP 反应不成功。这些发现表明, 氢氧化钠方法的基因组 DNA 制备是不兼容的 KASP. </li>
		<li> 使用分光光度计对基因组 DNA 进行量化。使用分子生物学等级水稀释基因组 DNA 模板, 使每个反应包含约 20-30 ng. 
		注: 量化4或5样本, 将它们汇集, 然后根据平均值为所有样品准备稀释。当使用这个协议为成人尾巴组织时, 1:100 稀释通常是充足的。不需要精确定量的 DNA 浓度. </li>
		<li> 添加0.14 和 #181; l KASP 底漆混合和5和 #181; l KASP 主混合每样品在冰到1.5 毫升离心管和混合好之前简要离心收集的内容在底部的管. 
		注: KASP 主混合是热和轻敏感, 保持在冰上的库存和避免直接光. </li>
		<li> 吸管5和 #181; 将底漆/主混合液的 L 置于96阱光学 pcr 板的井中, 适用于 real-time pcr 机的使用. </li>
		<li> 吸管5和 #181; 稀释 (约 5-7 ng/和 #181; l) DNA 模板样本进入每一个井和吸气吸管混合. </li>
		<li> 添加5和 #181; 核酸3口井为 no-template 控制 (NTC), 并添加5和 #181; 经稀释证实的纯合型野生类型、杂和纯合性突变 DNA 模板用于阳性对照. </li>
		<li> 在板上放置一个光学清晰的胶片密封, 确保胶片完全密封在每个井上. </li>
		<li> 简要地离心板材在 600 x g 收集底部的内容和从混合物中除去气泡. 
		注意: 把盘子放在冰上, 然后从光罩上, 直到准备继续. </li>
		<li> 使用显示在 表1中的循环程序 执行主 KASP 反应. </li>
		<li> 查看由分析计算机生成的结果散点图。一个成功的反应将显示4不同的点分组的情节 ( 图 3 ): 三样本组对应的基因型, 和一个分组的非贸易附近的起源。正控制应与适当的样本分组分隔. </li>
		<li> 如果计算机程序不识别与特定基因型相对应的绘图分组, 则可能需要附加的循环集 ( 表 2 )。重复该附加程序, 直到计算机通过基因型识别紧密分组. </li>
		<li> 将基因分型结果导出到电子表格文件中, 以便更方便地使用和分析. </li>
	</ol> </li> <li> 将已识别的杂动物放在主水系统上. </li> </ol>
 2. 第一轮子代评分 <ol> <li> 配对交叉同级子 <ol> 在动物从鳍片中恢复后, 设置成对的 intercrosses。使用分隔线确保胚胎是同步的 16 . </li>
		<li> 收集胚胎并将双亲对隔离在饲养笼中. </li>
		<li> 监视隔离的成人, 使攻击性鱼类可以被分离或提供覆盖 (鱼网丝很好地工作)。在静态水中, 鱼应该被喂食, 它们的水会随 IACUC 的指引而改变. </li>
	</ol> </li> <li> 胚胎饲养 <ol> <li> 阶段, 并在标准条件下将斑马鱼胚胎提升为幼虫 17 。
		<li> 收集型野生型动物使用玻璃吸管在5或6天, 当游泳膀胱在75% 的离合器充气。在育儿记录中放置表型的野生类型, 父母将其产生. 
		注: 与突变等位基因的隐性致死, 这是真实的许多骨骼突变, 合合突变体无法形成鱼鳔 18 , 19 。因此, 表型野生类型可以很容易地从他们的变种兄弟姐妹根据充气鱼鳔. </li>
	</ol> </li> <li> 新蓝/茜素红软骨和骨染色 <ol> <li> 麻醉幼虫在胚胎介质中使用0.17 克/升 Tricaine, 不充气其鱼鳔。收集动物到1.5 毫升离心管与宽口径玻璃巴斯德吸管。每管增加不超过100只幼虫. </li>
		<li> 从每管中取出胚胎水, 并在每管1毫升的2% 甲醛中修复动物. 
		 警告: 水中的粉煤灰是易燃的, 会引起严重的皮肤和眼部刺激。穿戴适当的个人防护设备, 如手套和眼睛保护, 使用后彻底洗手. </li>
		<li> 岩石为1小时, 较长的固定会损害骨染色。检查管定期在这和所有随后的摇摆步骤, 以确保没有幼虫已成为粘在一侧或成群在底部的管. </li>
		<li> 用玻璃吸管将定影液从试管中取出, 加入1毫升的50% 乙醇, 岩石10分钟. </li>
		<li> 准备新鲜染色液, 加入20和 #181; 每1毫升的新预混料 (0.04% 新 Blue/10 mM 氯化镁 2 /80% 乙醇) 的0.5% 茜素红。去除50% 乙醇, 加入1毫升的染色液, 每管, 和摇滚过夜。幼虫可在污渍中停留5天. </li>
		<li> 从管道中取出着色液, 并在每个管中添加1毫升 80% ethanol/10 mM 氯化镁 2 解决方案。岩石的管至少1小时;隔夜冲洗可以产生更清晰的新蓝色污渍. </li>
		<li> 从管中卸下 80% ethanol/10 mM 氯化镁 2 解决方案, 并添加1毫升的50% 乙醇; 岩石为 5 min. </li>
		<li> 从试管中取出50% 乙醇溶液, 并在5分钟内加入1毫升的25% 乙醇和岩石. </li>
		<li> 从管道中取出50% 乙醇溶液并添加1毫升新鲜准备的 3% H 2 O 2/0.5% KOH 漂白溶液。把瓶盖打开, 让管子在微管架中站立大约10分钟, 或者直到溶液上部可以看到微弱的褐色. 
		注: 溶液顶部形成一层气泡。在这一步需要仔细的定时, 因为过度漂白会导致一个糟糕的双重污点. </li>
		<li> 除去漂白溶液, 并添加1毫升25% 甘油/0.1% KOH; 岩石为 10 min 到 1 h. </li>
		<li> 从试管中取出25% 甘油/0.1% koh 溶液, 在每根管子上加1毫升的50% 甘油/0.1% koh 溶液, 在一夜之间将岩石移动10分钟. </li>
		<li> 从试管中取出50% 甘油/0.1% koh 溶液, 再在每根管子上加入50% 甘油/0.1% koh 溶液。隔夜摇摆将有助于消除气泡, 可以积累在幼虫。在未使用时, 将染色的骨骼准备工作保存在4和 #176; C. </li>
	</ol> </li> <li> 表型评分 <ol> <li> 为所选表型的显染色突变后代。在尼科尔斯 et al. 1 突变体对任何异位骨的发生都有评分. 
		注: 由于斑马鱼的父母是在静态水和茜素红染色可以褪色的时间, 这是重要的是要在几天内完成骨骼准备的骨骼. </li>
		<li> 显是具有表型的基因型的比例。按以下公式计算显百分比: % 显 = (具有表型的突变体)/(变种人总数) 和 #215; 100 </li> </ol> </li> </ol> 3。家庭选择 <ol> <li> 为下一代选择两个高显和两个低显家庭。除了显, 在选择繁殖的家庭时, 重要的是要考虑生育力和活力;有关此问题的详细信息, 请参见 讨论 . </li>
	<li> 给每个双亲配对一个子股票标识符:01,. 02, 等 </li>
	<li> 在主系统上的家庭双亲对有几个混合性和 #39; 同伴和 #39; 鱼。同伴鱼可以是任何斑马鱼线具有永久性的, 明显的区别性特征, 如改变色素沉着或鳍结构. 
	注: 虽然许多研究人员成功地只饲养了对在坦克, 有利结果被看见由几个伴生的鱼的住房对。这一可选步骤允许饲养的动物对在较大的学校饲养, 并容易检索, 以便他们可以反复交叉的需要。父母对可以反复交叉, 产生大的 full-sibling 家庭;请至少等待一周, 然后再重复过同一家长对. </li>
	<li> 从步骤2.2.2 中剔除幼虫系列. 这不是为下一代选择的. </li>
	<li> 从选定的家族中抽取含有野生型同胞幼虫的显, 从他们的变种兄弟姐妹中挑选, 并将其提升为正常的成年. </li>
	<li> 下一代将至少有四 full-sibling 家庭, 两个为上行线, 两个为下行线. </li>
	<li> 当幼虫达到性成熟度时, 从第一步开始与新的世代重复该协议. </li>
</ol>

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B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=barx1+represses+joints+and+promotes+cartilage+in+the+craniofacial+skeleton.">barx1 represses joints and promotes cartilage in the craniofacial skeleton.</a> Development. 140 (13), 2765-2775 (2013).</li><li>Sasaki, M. M., Nichols, J. T., Kimmel, C. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=edn1+and+hand2+Interact+in+early+regulation+of+pharyngeal+arch+outgrowth+during+zebrafish+development.">edn1 and hand2 Interact in early regulation of pharyngeal arch outgrowth during zebrafish development.</a> PLoS One. 8 (6), e67522 (2013).</li></ol>

作者没有什么可透露的。

选择性育种揭开基因功能的微妙之处
通过选择性育种, 改变突变型的表现形式或多或少是严重的, 这是获得基因功能新见解的直接途径。与未选定育种的标准方法相比, 本文所提出的协议可以更全面地了解突变表型。具体来说, 通过产生严重的菌株, 突变型的全广度可能会被揭示, 包括一些观测与未选定的股票。因此, 研究中的基因的新的发展作用可以被发现。相反,...

斑马鱼是一个强大的模型系统, 了解骨骼的发展。与变种斑马鱼菌株, 生物学家可以破译基因功能在骨化。然而, 斑马鱼骨骼突变表型可以出现可变显1,2,3,4 , 这可能会阻碍发展和遗传分析。这种方法的目的是三重的。首先, 生成斑马鱼突变线, 持续产生严重的表型, 使下游的发展研究, 如延时记录5和移植6。这类研究可能会因为试图研究表型而被削弱, 而这只是表现出不一致。其次, 近亲繁殖的斑马鱼菌株可以减少遗传背景的变化, 从而促进实验的一致性和重现性。例如, 对一个选择性的近亲繁殖菌株进行所有的原位杂交分析, 可以减少混杂的变异性并加强结论。第三, 产生严重的和轻度的菌株将揭示出整个表型系列, 可能产生的一个特定的突变。
乍一看, 选择性繁殖致命突变体似乎是不可能的。当被选入的动物死了的时候, 你怎么能为显繁殖呢？幸运的是, 选择育种的方法, 特别是子代试验, 在家畜育种项目中已经证明了许多年的有效性7,8。这些程序主要用于选择性育种的性状, 只存在于一个性别, 如牛奶生产的奶牛或鸡蛋生产母鸡。这些物种的雄性不能直接得分, 但他们的后代得分, 然后将价值分配给父母。从这一策略中, 这里所提出的协议包括对斑马鱼的固定和染色的突变后代进行评分, 这两个杂是为了一个感兴趣的突变基因。在决定哪些个体会产生下一代的时候, 显的一个表型会被分配给父母。我们发现这种方法是一种有效的方式转移显在斑马鱼致死性骨骼突变体1。
与其他研究类似, 这一选择育种协议考虑的标准, 如离合器大小, 后代的生存, 胚胎的正常发育和性别比例9。然而, 这些因素都是在一个突变背景下考虑的, 目的是转移突变显。因此, 该协议扩展了以往的选择育种范式, 提供了一种加强发育突变体分析和增加背景均匀性的方法。
这项协议需要广泛的基因分型, 所以必须事先制定一个可靠的快速基因分型协议。有许多基因分型协议可用10,11, 但是我们发现 KASP 基因分型12,13,14的速度更快, 成本效率更高, 比方法更可靠基于限制性酶裂解的扩增序列10。因此, 我们在这项工作中包括了一个 KASP 协议。此外, 我们的重点是在这个协议的骨骼突变表型, 并包括一个过程的新蓝/茜素红染色修改以前的协议15。
这里描述的方法是一个简单的策略, 转移致死突变显向上或向下。虽然这个协议的重点是骨骼突变的表型, 我们相信这将是一个有用的战略, 畜牧业的所有变种斑马鱼线。事实上, 这种育种策略的效用可能超越斑马鱼。我们预测, 这一协议可以修改, 以转移显在一个广泛的有机体范围。通过子代测试转移致死显可以帮助推动任何发展遗传学家的进步。

<table><tbody><tr><td>Paraformaldehyde, pelleted, solid</td><td>Ted Pella Co.</td><td>18501</td><td>Pelleted PFA is a safer alternative to powdered PFA</td></tr><tr><td>Magnesium Chloride, solid</td><td>Acros Organics</td><td>223210010</td><td></td></tr><tr><td>10x PBS, Aqueous</td><td>Fisher</td><td>BP3994</td><td></td></tr><tr><td>190 proof Ethanol</td><td></td><td></td><td></td></tr><tr><td>Alcian Blue, solid</td><td>Anatech Ltd.</td><td>867</td><td>Must be from Anatech</td></tr><tr><td>Alizarin Red, solid</td><td>Sigma</td><td>A5533-25G</td><td></td></tr><tr><td>Glycerol, liquid</td><td>Fisher</td><td>BP229 1</td><td></td></tr><tr><td>Hydrogen peroxide, liquid</td><td>Fisher</td><td>BP263500</td><td></td></tr><tr><td>Potassium hydroxide,&amp;nbsp; solid</td><td>Fisher</td><td>P250 500</td><td></td></tr><tr><td>StepOnePlus Real-time PCR Machine</td><td>Applied Biosystems</td><td></td><td></td></tr><tr><td>MicroAmp Fast Optical 96-well Reaction Plate with Barcode (0.1 mL)</td><td>Applied Biosystems</td><td>4346906</td><td></td></tr><tr><td>Microseal &#039;B&#039; seal</td><td>BioRad</td><td>MSB1001</td><td></td></tr><tr><td>KASP Master Mix, High ROX</td><td>LGC</td><td>KBS-1016-022</td><td>https://www.lgcgroup.com/products/kasp-genotyping-chemistry/#.WOPX41UrKUk</td></tr><tr><td>KASP By Design Primer Mix</td><td>LGC</td><td>KBS-2100-100</td><td></td></tr><tr><td>Tris HCl, solid</td><td>Fisher</td><td>BP153 500</td><td></td></tr><tr><td>potassium chloride, solid</td><td>Fisher</td><td>BP366 500</td><td></td></tr><tr><td>Tween-20, liquid</td><td>Fisher</td><td>BP337 100</td><td></td></tr><tr><td>Nonidet P40</td><td>ThermoFisher</td><td>28324</td><td></td></tr><tr><td>Tricaine-S</td><td>Western Chemicals</td><td></td><td></td></tr><tr><td>Proteinase K</td><td>Fisher</td><td>BP1700 100</td><td></td></tr><tr><td>T100 Thermal Cycler</td><td>BioRad</td><td>1861096</td><td></td></tr><tr><td>Controlled Drop Pasteur Pipets</td><td>Fisher</td><td>13-678-30</td><td></td></tr><tr><td>Nanodrop</td><td>ThermoFisher</td><td></td><td>for DNA quantitation</td></tr></tbody></table>

shifting zebrafish lethal skeletal mutant penetrance by progeny testing

斑马鱼变种的表型往往是不完全渗透, 只是在一些变种的显着。有趣的表型, 不一致的出现可能是很难研究, 并可能导致混淆的结果。这里描述的协议是一个简单的繁殖范例, 以增加和减少显在致命的斑马鱼骨骼突变体。由于致命突变体不能直接繁殖, 因此采用了典型的子代检测育种策略。该方法还包括 Kompetitive 等位基因特异性 PCR (KASP) 基因分型斑马鱼和染色幼虫斑马鱼软骨和骨的协议。应用这里描述的畜牧业战略可以增加一个有趣的骨骼表型的显, 使下游应用更具可重现性的结果。此外, 通过这一选择育种策略减少突变显, 可以揭示最关键的发展过程, 需要突变基因的功能。虽然骨骼是在这里被特别考虑, 我们建议, 这一方法将是有用的所有斑马鱼突变线。

该协议是一种长期饲养技术, 有助于了解斑马鱼骨骼突变体 (图 1)。后代测试的选择育种应该在几代人的显中产生一个整体的向下和向上的转变 (图 2)。在我们以前的工作中, 两轮的选择性育种使平均显向下, 从17% 到 3%1。同样, 在我们的上行线, 我们把平均显从63% 代上升到93% 在三世代。如果在四轮的选择育种后,...

Watch this Scientific Journal Video about Shifting Zebrafish Lethal Skeletal Mutant Penetrance by Progeny Testing at JoVE.com

Shifting Zebrafish Lethal Skeletal Mutant Penetrance by Progeny Testing

本议定书的目的是改变显致命的骨骼突变体表型在斑马鱼的选择性育种。致命的突变体不能成长为成年和繁殖, 因此本协议描述了通过后代测试来跟踪和选择显的方法。

通过子代检测改变斑马鱼致死性骨骼突变显

Zebrafish mutant phenotypes are often incompletely penetrant, only manifesting in some mutants. Interesting phenotypes that inconsistently appear can be ...

shifting-zebrafish-lethal-skeletal-mutant-penetrance-progeny

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Molecular Biology

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Mendelian Genetics

SCIENTISTS IN ACTION

7.5K 次观看.  University of Colorado Anschutz Medical Campus. 本议定书的目的是改变显致命的骨骼突变体表型在斑马鱼的选择性育种。致命的突变体不能成长为成年和繁殖, 因此本协议描述了通过后代测试来跟踪和选择显的方法。

视频: Shifting Zebrafish Lethal Skeletal Mutant Penetrance by Progeny Testing

Efficient Production and Identification of CRISPR/Cas9-generated Gene Knockouts in the Model System Danio rerio

Characterization of the clustered, regularly interspaced, short, palindromic repeat (CRISPR) system of Streptococcus pyogenes has enabled the development of a customizable platform to rapidly generate gene modifications in a wide variety of organisms, including zebrafish. CRISPR-based genome editing uses a single guide RNA (sgRNA) to target a CRISPR-associated (Cas) endonuclease to a genomic DNA (gDNA) target of interest, where the Cas endonuclease generates a double-strand break (DSB). Repair of DSBs by error-prone mechanisms lead to insertions and/or deletions (indels). This can cause frameshift mutations that often introduce a premature stop codon within the coding sequence, thus creating a protein-null allele. CRISPR-based genome engineering requires only a few molecular components and is easily introduced into zebrafish embryos by microinjection. This protocol describes the methods used to generate CRISPR reagents for zebrafish microinjection and to identify fish exhibiting germline transmission of CRISPR-modified genes. These methods include in vitro transcription of sgRNAs, microinjection of CRISPR reagents, identification of indels induced at the target site using a PCR-based method called a heteroduplex mobility assay (HMA), and characterization of the indels using both a low throughput and a powerful next-generation sequencing (NGS)-based approach that can analyze multiple PCR products collected from heterozygous fish. This protocol is streamlined to minimize both the number of fish required and the types of equipment needed to perform the analyses. Furthermore, this protocol is designed to be amenable for use by laboratory personal of all levels of experience including undergraduates, enabling this powerful tool to be economically employed by any research group interested in performing CRISPR-based genomic modification in zebrafish.

Efficient Production and Identification of CRISPR/Cas9-generated Gene Knockouts in the Model System Danio rerio

Targeted genome editing in the model system Danio rerio (zebrafish) has been greatly facilitated by the emergence of CRISPR-based approaches. Herein, we describe a streamlined, robust protocol for generation and identification of CRISPR-derived nonsense alleles that incorporates the heteroduplex mobility assay and identification of mutations using next-generation sequencing.

斑马斑马模型系统中 CRISPR/Cas9-generated 基因挖空的高效生产与鉴定

Characterization of the clustered, regularly interspaced, short, palindromic repeat (CRISPR) system of Streptococcus pyogenes has enabled the development ...

科研

遗传学

High-throughput DNA Extraction and Genotyping of 3dpf Zebrafish Larvae by Fin Clipping

Zebrafish (Danio rerio) possess orthologues for 84% of the genes known to be associated with human diseases. In addition, these animals have a short generation time, are easy to handle, display a high reproductive rate, low cost, and are easily amenable to genetic manipulations by microinjection of DNA in embryos. Recent advances in gene editing tools are enabling precise introduction of mutations and transgenes in zebrafish. Disease modeling in zebrafish often leads to larval phenotypes and early death which can be challenging to interpret if genotypes are unknown. This early identification of genotypes is also needed in experiments requiring sample pooling, such as in gene expression or mass spectrometry studies. However, extensive genotypic screening is limited by traditional methods, which in most labs are performed only on adult zebrafish or in postmortem larvae. We addressed this problem by adapting a method for the isolation of PCR-ready genomic DNA from live zebrafish larvae that can be achieved as early as 72 h post-fertilization (hpf). This time and cost-effective technique, improved from a previously published genotyping protocol, allows the identification of genotypes from microscopic fin biopsies. The fins quickly regenerate as the larvae develop. Researchers are then able to select and raise the desired genotypes to adulthood by utilizing this high-throughput PCR-based genotyping procedure.

Zebrafish have been used as reliable genetic model organisms in biomedical research, especially with the advent of gene-editing technologies. When larval phenotypes are expected, DNA extraction and genotype identification can be challenging. Here, we describe an efficient genotyping procedure for zebrafish larvae, by tail clipping, as early as 72-h post-fertilization.

3dpf 斑马鱼幼虫的高通量 DNA 提取及基因分型研究

Zebrafish (Danio rerio) possess orthologues for 84% of the genes known to be associated with human diseases. In addition, these animals have a short ...

Zebrafish Model of Neuroblastoma Metastasis

Zebrafish has emerged as an important animal model to study human diseases, especially cancer. Along with the robust transgenic and genome editing technologies applied in zebrafish modeling, the ease of maintenance, high-yield productivity, and powerful live imaging altogether make the zebrafish a valuable model system to study metastasis and cellular and molecular bases underlying this process in vivo. The first zebrafish neuroblastoma (NB) model of metastasis was developed by overexpressing two oncogenes, MYCN and LMO1, under control of the dopamine-beta-hydroxylase (d&#946;h) promoter. Co-overexpressed MYCN and LMO1 led to the reduced latency and increased penetrance of neuroblastomagenesis, as well as accelerated distant metastasis of tumor cells. This new model reliably reiterates many key features of human metastatic NB, including involvement of clinically relevant and metastasis-associated genetic alterations; natural and spontaneous development of metastasis in vivo; and conserved sites of metastases. Therefore, the zebrafish model possesses unique advantages to dissect the complex process of tumor metastasis in vivo.

This paper introduces the method of developing, characterizing, and tracking in real-time the tumor metastasis in zebrafish model of neuroblastoma, specifically in the transgenic zebrafish line with overexpression of MYCN and LMO1, which develops metastasis spontaneously.

斑马鱼神经母细胞瘤转移模型

Zebrafish has emerged as an important animal model to study human diseases, especially cancer. Along with the robust transgenic and genome editing ...

癌症研究

Screening Sperm for the Rapid Isolation of Germline Edits in Zebrafish

The advent of targeted CRISPR-Cas nuclease technologies has revolutionized the ability to perform precise genome editing in both established and emerging model systems. CRISPR-Cas genome editing systems use a synthetic guide RNA (sgRNA) to target a CRISPR-associated (Cas) endonuclease to specific genomic DNA loci, where the Cas endonuclease generates a double-strand break. The repair of double-strand breaks by intrinsic error-prone mechanisms leads to insertions and/or deletions, disrupting the locus. Alternatively, the inclusion of double-stranded DNA donors or single-stranded DNA oligonucleotides in this process can elicit the inclusion of precise genome edits ranging from single nucleotide polymorphisms to small immunological tags or even large fluorescent protein constructs. However, a major bottleneck in this procedure can be finding and isolating the desired edit in the germline. This protocol outlines a robust method for screening and isolating germline mutations at specific loci in Danio rerio (zebrafish); however, these principles may be adaptable in any model where in vivo sperm collection is possible.

CRISPR-Cas technologies have revolutionized the field of genome editing. However, finding and isolating the desired germline edit remains a major bottleneck. Therefore, this protocol describes a robust method for quickly screening F0 CRISPR-injected zebrafish sperm for germline edits using standard PCR, restriction digest, and gel electrophoresis techniques.

筛选精子以快速分离斑马鱼的种系编辑

The advent of targeted CRISPR-Cas nuclease technologies has revolutionized the ability to perform precise genome editing in both established and emerging ...

Genotyping and Quantification of In Situ Hybridization Staining in Zebrafish

In situ hybridization (ISH) is an important technique that enables researchers to study mRNA distribution in situ and has been a critical technique in developmental biology for decades. Traditionally, most gene expression studies relied on visual evaluation of the ISH signal, a method that is prone to bias, particularly in cases where sample identities are known a priori. We have previously reported on a method to circumvent this bias and provide a more accurate quantification of ISH signals. Here, we present a simple guide to apply this method to quantify the expression levels of genes of interest in ISH-stained embryos and correlate that with their corresponding genotypes. The method is particularly useful to quantify spatially restricted gene expression signals in samples of mixed genotypes and it provides an unbiased and accurate alternative to the traditional visual scoring methods.

Gene editing technologies have enabled researchers to generate zebrafish mutants to investigate gene function with relative ease. Here, we provide a guide to perform parallel embryo genotyping and quantification of in situ hybridization signals in zebrafish. This unbiased approach provides greater accuracy in phenotypical analyses based on in situ hybridization.

斑马鱼原位杂交染色的基因分型与定量

In situ hybridization (ISH) is an important technique that enables researchers to study mRNA distribution in situ and has been a critical technique in ...

发育生物学

Examining Muscle Regeneration in Zebrafish Models of Muscle Disease

Skeletal muscle has a remarkable ability to regenerate following injury, which is driven by obligate tissue resident muscle stem cells. Following injury, the muscle stem cell is activated and undergoes cell proliferation to generate a pool of myoblasts, which subsequently differentiate to form new muscle fibers. In many muscle wasting conditions, including muscular dystrophy and ageing, this process is impaired resulting in the inability of muscle to regenerate. The process of muscle regeneration in zebrafish is highly conserved with mammalian systems providing an excellent system to study muscle stem cell function and regeneration, in muscle wasting conditions such as muscular dystrophy. Here, we present a method to examine muscle regeneration in zebrafish models of muscle disease. The first step involves the use of a genotyping platform that allows the determination of the genotype of the larvae prior to eliciting an injury. Having determined the genotype, the muscle is injured using a needle stab, following which polarizing light microscopy is used to determine the extent of muscle regeneration. We therefore provide a high throughput pipeline which allows the examination of muscle regeneration in zebrafish models of muscle disease.

Skeletal muscle regeneration is driven by tissue resident muscle stem cells, which are impaired in many muscle diseases such as muscular dystrophy, and this results in the inability of muscle to regenerate. Here, we describe a protocol that allows the examination of muscle regeneration in zebrafish models of muscle disease.

检查斑马鱼肌肉疾病模型中的肌肉再生

Skeletal muscle has a remarkable ability to regenerate following injury, which is driven by obligate tissue resident muscle stem cells. Following injury, ...

In Vivo Modeling of the Morbid Human Genome using Danio rerio

Here, we present methods for the development of assays to query potentially clinically significant nonsynonymous changes using in vivo complementation in zebrafish. Zebrafish (Danio rerio) are a useful animal system due to their experimental tractability; embryos are transparent to enable facile viewing, undergo rapid development ex vivo, and can be genetically manipulated.1 These aspects have allowed for significant advances in the analysis of embryogenesis, molecular processes, and morphogenetic signaling. Taken together, the advantages of this vertebrate model make zebrafish highly amenable to modeling the developmental defects in pediatric disease, and in some cases, adult-onset disorders. Because the zebrafish genome is highly conserved with that of humans (~70% orthologous), it is possible to recapitulate human disease states in zebrafish. This is accomplished either through the injection of mutant human mRNA to induce dominant negative or gain of function alleles, or utilization of morpholino (MO) antisense oligonucleotides to suppress genes to mimic loss of function variants. Through complementation of MO-induced phenotypes with capped human mRNA, our approach enables the interpretation of the deleterious effect of mutations on human protein sequence based on the ability of mutant mRNA to rescue a measurable, physiologically relevant phenotype. Modeling of the human disease alleles occurs through microinjection of zebrafish embryos with MO and/or human mRNA at the 1-4 cell stage, and phenotyping up to seven days post fertilization (dpf). This general strategy can be extended to a wide range of disease phenotypes, as demonstrated in the following protocol. We present our established models for morphogenetic signaling, craniofacial, cardiac, vascular integrity, renal function, and skeletal muscle disorder phenotypes, as well as others.

In Vivo Modeling of the Morbid Human Genome using Danio rerio

Here, we present a systematic approach for developing physiologically relevant, sensitive and specific in vivo assays for interpreting variation in human pathology. Transient genetic manipulation via microinjection of WT and mutant human mRNA and morpholino (MO) antisense oligonucleotides harness the tractability of the developing zebrafish embryo to rapidly assay pathogenic mutations, especially, but not exclusively, in the context of human developmental disorders.

在体内病态的人类基因组建模使用斑马鱼

Here,  we present methods for the development of assays to query potentially clinically  significant nonsynonymous changes using in  vivo complementation ...

生物学

Analysis of Skeletal Muscle Defects in Larval Zebrafish by Birefringence and Touch-evoke Escape Response Assays

Zebrafish (Danio rerio) have become a particularly effective tool for modeling human diseases affecting skeletal muscle, including muscular dystrophies1-3, congenital myopathies4,5, and disruptions in sarcomeric assembly6,7, due to high genomic and structural conservation with mammals8. Muscular disorganization and locomotive impairment can be quickly assessed in the zebrafish over the first few days post-fertilization. Two assays to help characterize skeletal muscle defects in zebrafish are birefringence (structural) and touch-evoked escape response (behavioral).
Birefringence is a physical property in which light is rotated as it passes through ordered matter, such as the pseudo-crystalline array of muscle sarcomeres9. It is a simple, noninvasive approach to assess muscle integrity in translucent zebrafish larvae early in development. Wild-type zebrafish with highly organized skeletal muscle appear very bright amidst a dark background when visualized between two polarized light filters, whereas muscle mutants have birefringence patterns specific to the primary muscular disorder they model. Zebrafish modeling muscular dystrophies, diseases characterized by myofiber degeneration followed by repeated rounds of regeneration, exhibit degenerative dark patches in skeletal muscle under polarized light. Nondystrophic myopathies are not associated with necrosis or regenerative changes, but result in disorganized myofibers and skeletal muscle weakness. Myopathic zebrafish typically show an overall reduction in birefringence, reflecting the disorganization of sarcomeres.
The touch-evoked escape assay involves observing an embryo&#39;s swimming behavior in response to tactile stimulation10-12. In comparison to wild-type larvae, mutant larvae frequently display a weak escape contraction, followed by slow swimming or other type of impaired motion that fails to propel the larvae more than a short distance12. The advantage of these assays is that disease progression in the same fish type can be monitored in vivo for several days, and that large numbers of fish can be analyzed in a short time relative to higher vertebrates.

The zebrafish is now an established and powerful tool for modeling muscular dystrophies, congenital myopathies, and related neuromuscular diseases. Birefringence and touch-evoked escape behavior are two common noninvasive assays used to determine the degree of muscular disorganization and locomotive impairment of zebrafish embryos during early development.

Zebrafish (Danio rerio) have become a particularly effective tool for modeling human diseases affecting skeletal muscle, including muscular ...

Production of Haploid Zebrafish Embryos by In Vitro Fertilization

The zebrafish has become a mainstream vertebrate model that is relevant for many disciplines of scientific study. Zebrafish are especially well suited for forward genetic analysis of developmental processes due to their external fertilization, embryonic size, rapid ontogeny, and optical clarity &#8211; a constellation of traits that enable the direct observation of events ranging from gastrulation to organogenesis with a basic stereomicroscope. Further, zebrafish embryos can survive for several days in the haploid state. The production of haploid embryos in vitro is a powerful tool for mutational analysis, as it enables the identification of recessive mutant alleles present in first generation (F1) female carriers following mutagenesis in the parental (P) generation. This approach eliminates the necessity to raise multiple generations (F2, F3, etc.) which involves breeding of mutant families, thus saving the researcher time along with reducing the needs for zebrafish colony space, labor, and the husbandry costs. Although zebrafish have been used to conduct forward screens for the past several decades, there has been a steady expansion of transgenic and genome editing tools. These tools now offer a plethora of ways to create nuanced assays for next generation screens that can be used to further dissect the gene regulatory networks that drive vertebrate ontogeny. Here, we describe how to prepare haploid zebrafish embryos. This protocol can be implemented for novel future haploid screens, such as in enhancer and suppressor screens, to address the mechanisms of development for a broad number of processes and tissues that form during early embryonic stages.

Production of Haploid Zebrafish Embryos by In Vitro Fertilization

The zebrafish is a powerful model system for developmental biology and human disease research due to their genetic similarity with higher vertebrates. This protocol describes a methodology to create haploid zebrafish embryos that can be utilized for forward screen strategies to identify recessive mutations in genes essential for early embryogenesis.

生产单倍体斑马鱼的胚胎通过<em&gt;体外</em&gt;施肥

The zebrafish has become a mainstream vertebrate model that is relevant for many disciplines of scientific study. Zebrafish are especially well suited for ...

Efficient PAM-Less Base Editing for Zebrafish Modeling of Human Genetic Disease with zSpRY-ABE8e

CRISPR/Cas9 technology has increased the value of zebrafish for modeling human genetic diseases, studying disease pathogenesis, and drug screening, but protospacer adjacent motif (PAM) limitations are a major obstacle to creating accurate animal models of human genetic disorders caused by single-nucleotide variants (SNVs). Until now, some SpCas9 variants with broad PAM compatibility have shown efficiency in zebrafish. The application of the optimized SpRY-mediated adenine base editor (ABE), zSpRY-ABE8e, and synthetically modified gRNA in zebrafish has enabled efficient adenine-guanine base conversion without PAM restriction. Described here is a protocol for efficient adenine base editing without PAM limitation in zebrafish using zSpRY-ABE8e. By injecting a mixture of zSpRY-ABE8e mRNA and synthetically modified gRNA into zebrafish embryos, a zebrafish disease model was constructed with a precise mutation that simulated a pathogenic site of the TSR2 ribosome maturation factor (tsr2). This method provides a valuable tool for the establishment of accurate disease models for studying disease mechanisms and treatments.

This protocol describes how to perform efficient adenine base editing without PAM limitation to construct a precise zebrafish disease model using zSpRY-ABE8e.

使用 zSpRY-ABE8e 对斑马鱼进行人类遗传病建模的高效无 PAM 碱基编辑

CRISPR/Cas9 technology has increased the value of zebrafish for modeling human genetic diseases, studying disease pathogenesis, and drug screening, but ...