Name: high-throughput dna extraction and genotyping of 3dpf zebrafish larvae by fin clipping
Uploaded: 2018-06-29T15:00:00.000+00:00
Duration: 10 min 12 s
Description: Watch this Scientific Journal Video about High-throughput DNA Extraction and Genotyping of 3dpf Zebrafish Larvae by Fin Clipping at JoVE.com

作者希望感谢稀有疾病模型和机制 (RDMM) 网络 (由加拿大卫生研究所 (卫生研究院) 和加拿大基因组资助)。CK 获得 UROP 奖学金 (渥太华大学) 的支持。DLJ 得到加拿大 Vanier 研究生奖学金的支持。由加拿大卫生研究院 (卫生研究院) 博士后奖学金奖资助。作者感谢美国遗传学学会批准发布这项协议, 并使用修改的补充图5的修订,等等。

有关动物的程序已获批准, 并符合加拿大动物保育委员会和渥太华大学动物保育委员会提供的动物保育指引。
1. 准备
<ol><li>通过用高压釜胶带在其内部表面上贴上9厘米的培养皿盖, 准备解剖表面。在立体声显微镜下定位。</li>
	<li>放置 3-5 天后受精 (dpf) 斑马鱼幼虫在培养皿和麻醉˜1.5 毫米 Tricaine 1x E3 胚介质 (5 毫米氯化钠, 0.17 毫米氯化钾, 0.33 毫米 CaCl2, 0.33 毫米 MgSO4, pH 7.2)。</li>
	<li>用剪刀或剃刀刀片切断 P1000 吸管尖端的末端, 直径为2毫米, 以容纳3的 dpf 幼虫, 压力最小。</li>
</ol>2. 斑马鱼幼虫的鳍片修剪
<ol><li>使用改良的 P1000 微提取麻醉幼虫, 将斑马鱼幼虫放在培养皿盖上放置的高压釜胶带上。</li>
	<li>用微去除幼虫周围的过量 Tricaine 溶液。该区域应尽可能干燥, 以成功的部分, 并拿起鳍, 但仍然湿, 以确保生存。</li>
	<li>使用微手术刀, 在一个立体声显微镜下的尾鳍切片, 如图 1所示, 如威尔金森et al16。在切口期间, 在尾翅的颜料间隙部位, 在血液循环的极限处应用稳定的下行压力 (图 1A)。施加温和的压力以避免损坏脊索。</li>
	<li>在显微镜下可视化切片鳍, 并将该片放置在 microscalpel 刀片尖端的顶端 (图 2A)。将含有鳍的 microscalpel 放在一小片滤纸的表面上。 注意: 由于在横断组织中存在黑色素细胞, 这一步骤允许将鳍可视化为一个小黑点 (图 2B)。</li>
	<li>使用剪刀和镊子 (图 2C), 将含有翅片的滤纸转移到一个96井 PCR 板上, 其中包含 25 ul 每井50毫米氢氧化钠溶液 (图 2D)。</li>
	<li>准备一个平底的96井板, 根据 PCR 板的标签对油井进行编号。使用改良的 P1000 吸管填充200µL 新鲜 1x E3 胚培养基, 小心收集斑马鱼幼虫使用温和的压力。将幼虫放在96井组织培养板中, 与 PCR 板相同的井数 (图 2E)。</li>
	<li>确保过滤器的纸张在溶液中被充分淹没 (图 2F)。清洁 microscapel 和镊子之间的刀片, 以防止交叉污染, 通过浸渍到70% 乙醇解决方案。用干净的纸巾擦拭刀片/擦拭。</li>
	<li>重复步骤 2.1-2.7, 直到所有的胚胎被修剪。</li>
	<li>将幼虫储存在96井板中28摄氏度, 直到鉴定出基因型。 注意: 胚胎介质不需要改变, 因为该协议可以很容易地完成在不到2天。如果需要更多时间, 建议进行媒体更改。</li>
</ol>3. 基因组 DNA 提取
<ol><li>密封96井 PCR 板和离心机的样品在 1000 x g 1 分钟, 以确保所有的过滤器纸被淹没在氢氧化钠溶液中。</li>
	<li>为组织裂解, 加热样品在一个 thermocycler 在95°c 为5分钟然后冷却到4°c 为10分钟。</li>
	<li>添加 6 ul 500 毫米三 HCl, pH 8.0 到每个样品。涡。</li>
	<li>在室温下, 将该板材离心为 1500 x g, 5 分钟。</li>
	<li>每个 PCR 反应使用 1.5 ul 的 DNA 上清。</li>
	<li>准备 PCR 的基因型的幼虫, 如表 1-2所示。 注: 本协议测试了两个应用: 多重 PCR 反应揭示的基因型使用琼脂糖凝胶9, 和 heteroduplex 熔融试验 (HMA) 揭示基因型的聚丙烯酰胺凝胶电泳 (页)17。</li>
</ol>4. 用螯合树脂提取替代基因组
<ol><li>完成步骤2.5 通过添加带修剪翅片的滤纸到96井 PCR 板, 其中含有 30 ul 5% 螯合树脂 (含配对 iminodiacetate 离子的苯乙烯-二乙烯苯共聚物)。 注: 这种树脂通常用于组织裂解和制备 PCR 准备好的 DNA 快速有效的方式18。</li>
	<li>按照指示执行步骤 2.6-2.8。</li>
	<li>密封96井 PCR 板, 并简要离心样品, 以确保所有过滤纸被淹没在螯合树脂内。</li>
	<li>为组织裂解, 加热样品在一个 thermocycler 在95°c 为15分钟然后冷却到4°c 为10分钟。 注意: 这一步允许在 DNA 和 RNA 保持溶液时, 将极性树脂珠子的溶解和随后的结合到细胞成分中。</li>
	<li>简要地离心样品颗粒树脂珠并且获取脱氧核糖核酸在悬浮。</li>
	<li>按照步骤 3.5-3.6 完成基因分型程序。</li>
</ol>5. 应用 1: 多重 PCR 后琼脂糖凝胶分析
<ol><li>下表 1, 建立了多重 PCR 反应, 以使纯合突变体, 杂合子和野生幼虫之间的区别。利用表 3中描述的循环条件, 使用96井热循环仪进行 PCR 反应。 注: pcr 反应也可以在任何其他常规 pcr 热循环仪进行。这个例子描述了使用的 PCR 策略. 9 ) 在同一复用反应中识别aldh7a1和 5-bp 插入突变等位基因。</li>
	<li>在硼酸钠缓冲液中制备1% 琼脂糖凝胶, 染色 1x GelRed。 注: 20x 硼酸钠缓冲液由40毫升10米氢氧化钠制成, 1800 毫升 ddH2O, pH 调整为 8.5, 硼酸76克, 然后用 ddH2O 完成2升最终容积。</li>
	<li>在1x 硼酸钠缓冲液中运行凝胶15分钟, 250 v。为方便这一过程, 如果可能的话, 使用与多通道滴管兼容的凝胶系统, 通过减少加载样本的时间来实现更高的吞吐量能力。</li>
	<li>图像的凝胶下紫外线 (UV) 光允许歧视的不同基因型。</li>
</ol>6. 应用 2: Heteroduplex 熔融试验
<ol><li>准备12% 页凝胶使用1.5 毫米间隔板和15样品梳。用12.21 毫升的 ddH2O、2.52 毫升10x 三/硼酸/EDTA ()、10毫升30% 丙烯酰胺、250硫酸铵 (APS) 和 10% tetramethylethylenediamine (TEMED) 的 ul 来制备两页凝胶。使用1x 缓冲器 (0.089 米三盐酸, 0.089 米硼酸和0.002 米乙二胺乙酸 (EDTA)) 作为运行缓冲器。</li>
	<li>将 PCR 产品加热94摄氏度, 5 分钟。</li>
	<li>冷却管在冰或在 thermocycler 10 分钟在4°c。</li>
	<li>每 PCR 样品负载 10 ul 和8个分子量标记 ul。</li>
	<li>使用垂直电泳系统1小时, 或直到分子量标记几乎达到玻璃板的脚线, 以1x 的150伏运行该页面。</li>
	<li>图像的凝胶在紫外线下, 以允许歧视的不同基因型。</li>
</ol>

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作者声明他们没有竞争的财政利益。

基因编辑工具已经被用来精确地引入突变和转基因在过去几年斑马鱼5,6,7,8。在斑马鱼进行疾病建模时, 早期幼虫或幼年表型通常观察到9、12、13、14。这里提出的协议描述了从小斑马鱼幼虫尾活检中提取 PC...

斑马鱼 (斑马斑马)是一种脊椎动物有机体, 广泛用作疾病调查的模型, 以及治疗假说1,2,3的临床前检验。这些动物有短的世代时间, 容易处理, 显示高繁殖率, 低费用, 并且容易地接受基因操作通过显微注射 DNA 在胚胎4。通过新的转基因和基因编辑技术的不断发展, 如锌指核酸 (ZFN)5, 核酸 (TALENs)6,7和聚集定期 Interspaced 短回文重复 (CRISPR)/CRISPR 相关 9 (Cas9) 系统8, 斑马鱼准备大大提高对几个病理条件的理解。这些技术已经被用来在斑马鱼的体细胞和生殖细胞中产生靶向性的挖空, 有效地培养转基因动物8。最近在文献中的例子包括成功地开发了癫痫的遗传模型和代谢紊乱在斑马鱼3,9,10,11,12.
随着斑马鱼基因组编辑的进展, 快速而可靠的基因分型已成为不可否认的限速步骤。在预测基因组编辑目标站点附近的特定 DNA 突变的识别是该协议的一个重要阶段。传统上, 鳍片修剪的基因分型技术通常只在青少年或成年斑马鱼身上进行。然而, 将斑马鱼饲养到成年 (> 2 月) 的时间大大延缓了研究的进展。在许多情况下, 由于关键基因的挖空 (例如疾病建模) 或功能突变导致的表型效应, 成年无法实现。此外, 一些化验需要收集幼虫, 如在 RNA 或代谢物提取, 从而涉及事先鉴定的基因型, 这可能是挑战时, 只有特殊的基因组技术可用。上述例子突出了幼虫基因分型技术的重要性, 同时也是精确的, 能够充分恢复和正常发育的幼虫。早期斑马鱼的基因分型目前似乎没有得到广泛应用;最近的一些疾病模型的出版物反映了这一点, 其中幼虫基因型是通过验尸后基因类型确定的, 而不是在实验12、13、14的执行之前分基因。本文提出了一种幼虫夹鳍策略, 旨在改进以前建立的基因分型程序。
过去, 采用蛋白酶 K 消解基因型活斑马鱼幼虫的策略导致了变聚合酶链反应 (PCR) 效率15。虽然最近公布的显微尾部活检技术提供了更有希望的结果16, 我们和其他小组无法重现的高效率和 DNA 恢复报告的作者。威尔金森等所描述的协议的一个重大倒退。16可能是尾巴组织的转移到 PCR 管。由于其显微尺寸, 很难确保鳍被正确收集和配发的吹打。为了解决这一障碍, 我们开发了一种改进的协议, 用于对大量活斑马鱼幼虫进行基因分型, 其效率为 100%9。使用 microscalpel, 鳍尾的尖端被移除并放置在一张滤纸上。该片含有组织的滤纸可以很容易地可视化, 并正确地放入 PCR 管。然后进行基因组 DNA 提取, 然后进行 PCR 扩增的感兴趣区域基因型的标本。该方法的优点有: PCR 效率高, 假阳性率低, 死亡率低。另外, 使用该协议可以对大量胚胎进行基因分型。本文所述的协议允许在 2-3 小时内用这种方法对数以百计的幼虫进行翅片修剪, 并在两天内正确识别 genotyped 鱼和尾再生。

<table><tbody><tr><td>Petri dish</td><td>Corning</td><td>430167</td><td></td></tr><tr><td>Microscalpel</td><td>World Precision Instruments</td><td>500249</td><td></td></tr><tr><td>Stereomicroscope</td><td>Nikon</td><td>SMZ1500</td><td></td></tr><tr><td>Tricaine/Ethyl 3-aminobenzoate methanesulfonate</td><td>Sigma-Aldrich</td><td>E10521</td><td></td></tr><tr><td>96-well Tissue-culture plate</td><td>Costar</td><td>3595</td><td></td></tr><tr><td>96-well PCR plate</td><td>Fisher</td><td>14230232</td><td></td></tr><tr><td>NaOH</td><td>Fisher</td><td>S25548A&amp;nbsp;</td><td></td></tr><tr><td>Tris base&amp;nbsp;</td><td>Sigma-Aldrich</td><td>T1503</td><td></td></tr><tr><td>NaCl</td><td>Fisher</td><td>BP358-10</td><td></td></tr><tr><td>KCl</td><td>Sigma-Aldrich</td><td>P3911</td><td></td></tr><tr><td>MgSO4</td><td>Sigma-Aldrich</td><td>230391</td><td></td></tr><tr><td>5% Chelex 100 sodium form</td><td>Sigma-Aldrich</td><td>C7901</td><td></td></tr><tr><td>GelRed 1x</td><td>Biotium</td><td>41003</td><td></td></tr><tr><td>Boric Acid</td><td>Sigma-Aldrich</td><td>B6768</td><td></td></tr><tr><td>Sub-Cell Model 192 system</td><td>Bio-Rad&amp;nbsp;</td><td>1704508</td><td></td></tr><tr><td>30 % Acrylamide/Bis Solution, 37.5: 1</td><td>Bio-Rad&amp;nbsp;</td><td>1610158</td><td></td></tr><tr><td>GoTaq Green Master Mix, 2X</td><td>Promega</td><td>M7123</td><td></td></tr><tr><td>paper wipes (Kimwipes&amp;reg; disposable wipers)</td><td>Sigma-Aldrich</td><td>Z188956</td><td></td></tr><tr><td>1kb-plus molecylar weight marker&amp;nbsp;</td><td>Thermo Scientific</td><td>SM1343</td><td></td></tr><tr><td>Nuclease free water&amp;nbsp;</td><td>Sigma-Aldrich</td><td>W4502</td><td></td></tr><tr><td>Sub-Cell Model 192 system (high-throughput agarose gel system)</td><td>Bio-Rad&amp;nbsp;</td><td>1704508</td><td></td></tr><tr><td>vertical gel electrophoresis system Mini-PROTEAN Tetra Cell&amp;nbsp;</td><td>Bio-Rad&amp;nbsp;</td><td>1658004</td><td></td></tr></tbody></table>

high-throughput dna extraction and genotyping of 3dpf zebrafish larvae by fin clipping

斑马鱼 (斑马斑马) 拥有 orthologues 已知与人类疾病相关的84% 的基因。此外, 这些动物有短的世代时间, 容易处理, 显示高繁殖率, 低成本, 并且容易地接受基因操作通过微注射 DNA 在胚胎。基因编辑工具的最新进展使斑马鱼的突变和转基因得到精确的引入。在斑马鱼的疾病建模往往导致幼虫表型和早期死亡, 这可能是挑战, 以解释, 如果基因的未知。在需要样本池的实验中也需要这种早期的基因型鉴定, 例如基因表达或质谱研究。然而, 广泛的基因型筛选受传统方法的限制, 在大多数实验室仅对成年斑马鱼或死后幼虫进行。我们解决这个问题的方法是, 采用一种分离 PCR 现成的基因组 DNA 从活斑马鱼幼虫, 可以达到早在72小时后受精 (hpf)。这一时间和成本效益的技术, 改进了从以前公布的基因分型协议, 允许鉴定的基因, 从微观鳍活检。随着幼虫的发育, 鳍迅速再生。然后, 研究人员可以利用这种高通量 PCR 的基因分型方法, 选择并提高所期望的成年期。

protocoll 的效率是通过基因分型的异型十字架 (aldh7a1+/ot100, 这里显示为aldh7a1+/) (图 3A-C)9。aldh7a1ot100突变等位基因有一个5基对 (bp) 插入的第一个编码外显子的斑马鱼aldh7a1基因导致 frameshift 和丧失功能, 由于早期停止密码子9。在多重反应中?...

Watch this Scientific Journal Video about High-throughput DNA Extraction and Genotyping of 3dpf Zebrafish Larvae by Fin Clipping at JoVE.com

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

3dpf 斑马鱼幼虫的高通量 DNA 提取及基因分型研究

斑马鱼在生物医学研究中被用作可靠的遗传模型有机体, 特别是随着基因编辑技术的出现。当幼虫表型预计, DNA 提取和基因的鉴定可能是挑战。在这里, 我们描述了一个有效的基因分型程序的斑马鱼幼虫, 通过尾部修剪, 早在72小时后受精。

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

high-throughput-dna-extraction-genotyping-3dpf-zebrafish-larvae-fin

Research

JoVE Journal

Genetics

13.9K 次观看.  Children's Hospital of Eastern Ontario Research Institute. 斑马鱼在生物医学研究中被用作可靠的遗传模型有机体, 特别是随着基因编辑技术的出现。当幼虫表型预计, DNA 提取和基因的鉴定可能是挑战。在这里, 我们描述了一个有效的基因分型程序的斑马鱼幼虫, 通过尾部修剪, 早在72小时后受精。

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

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

Profiling DNA Replication Timing Using Zebrafish as an In Vivo Model System

DNA replication timing is an important cellular characteristic, exhibiting significant relationships with chromatin structure, transcription, and DNA mutation rates. Changes in replication timing occur during development and in cancer, but the role replication timing plays in development and disease is not known. Zebrafish were recently established as an in vivo model system to study replication timing. Here is detailed the protocols for using the zebrafish to determine DNA replication timing. After sorting cells from embryos and adult zebrafish, high-resolution genome-wide DNA replication timing patterns can be constructed by determining changes in DNA copy number through analysis of next generation sequencing data. The zebrafish model system allows for evaluation of the replication timing changes that occur in vivo throughout development, and can also be used to assess changes in individual cell types, disease models, or mutant lines. These methods will enable studies investigating the mechanisms and determinants of replication timing establishment and maintenance during development, the role replication timing plays in mutations and tumorigenesis, and the effects of perturbing replication timing on development and disease.

Profiling DNA Replication Timing Using Zebrafish as an In Vivo Model System

Zebrafish were recently used as an in vivo model system to study DNA replication timing during development. Here is detailed the protocols for using zebrafish embryos to profile replication timing. This protocol can be easily adapted to study replication timing in mutants, individual cell types, disease models, and other species.

使用斑马鱼作为体内模型系统分析 DNA 复制定时

DNA replication timing is an important cellular characteristic, exhibiting significant relationships with chromatin structure, transcription, and DNA ...

Isolation and Characterization of Single Cells from Zebrafish Embryos

The zebrafish (Danio rerio) is a powerful model organism to study vertebrate development. Though many aspects of zebrafish embryonic development have been described at the morphological level, little is known about the molecular basis of cellular changes that occur as the organism develops. With recent advancements in microfluidics and multiplexing technologies, it is now possible to characterize gene expression in single cells. This allows for investigation of heterogeneity between individual cells of specific cell populations to identify and classify cell subtypes, characterize intermediate states that occur during cell differentiation, and explore differential cellular responses to stimuli. This study describes a protocol to isolate viable, single cells from zebrafish embryos for high throughput multiplexing assays. This method may be rapidly applied to any zebrafish embryonic cell type with fluorescent markers. An extension of this method may also be used in combination with high throughput sequencing technologies to fully characterize the transcriptome of single cells. As proof of principle, the relative abundance of cardiac differentiation markers was assessed in isolated, single cells derived from nkx2.5 positive cardiac progenitors. By evaluation of gene expression at the single cell level and at a single time point, the data support a model in which cardiac progenitors coexist with differentiating progeny. The method and work flow described here is broadly applicable to the zebrafish research community, requiring only a labeled transgenic fish line and access to microfluidics technologies.

This protocol describes a method for isolating single cells from zebrafish embryos, enriching for cells of interest, capturing zebrafish cells in microfluidic based single cell multiplex systems, and assessing gene expression from single cells.

分离和斑马鱼胚胎单细胞的表征

The zebrafish (Danio rerio) is a powerful model organism to study vertebrate development. Though many aspects of zebrafish embryonic development have been ...

发育生物学

Time-lapse Live Imaging of Clonally Related Neural Progenitor Cells in the Developing Zebrafish Forebrain

Precise patterns of division, migration and differentiation of neural progenitor cells are crucial for proper brain development and function1,2. To understand the behavior of neural progenitor cells in the complex in vivo environment, time-lapse live imaging of neural progenitor cells in an intact brain is critically required. In this video, we exploit the unique features of zebrafish embryos to visualize the development of forebrain neural progenitor cells in vivo. We use electroporation to genetically and sparsely label individual neural progenitor cells. Briefly, DNA constructs coding for fluorescent markers were injected into the forebrain ventricle of 22 hours post fertilization (hpf) zebrafish embryos and electric pulses were delivered immediately. Six hours later, the electroporated zebrafish embryos were mounted with low melting point agarose in glass bottom culture dishes. Fluorescently labeled neural progenitor cells were then imaged for 36hours with fixed intervals under a confocal microscope using water dipping objective lens. The present method provides a way to gain insights into the in vivo development of forebrain neural progenitor cells and can be applied to other parts of the central nervous system of the zebrafish embryo.

The present video demonstrates a method which takes advantage of the combination of electroporation and confocal microscopy to perform live imaging on individual neural progenitor cells in the developing zebrafish forebrain. In vivo analysis of the development of forebrain neural progenitor cells at a clonal level can be achieved in this way.

在发展中国家斑马鱼的前脑的神经祖细胞克隆相关的定时实时成像

Precise patterns of division, migration and differentiation of neural progenitor cells are crucial for proper brain development and function1,2. To ...

神经科学

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 ...

<meta charset="utf8"></head><body>使用 CRISPR/Cas9 敲入在斑马鱼幼虫中进行荧光蛋白标记

Fluorescence Labeling to Visualize Low-Expressed Proteins in Zebrafish

CRISPR/Cas9-mediated knock-in (KI) technology allows for easier fluorescent-protein tagging in zebrafish (Danio rerio), a preferred model organism for in vivo imaging due to its transparency during the early developmental stage. Here, we provide a detailed protocol for performing high-efficiency fluorescence gene KI, rapid screening for KI founders, and low-abundance protein tracing in zebrafish larvae, which will lay a critical foundation for subsequent physio-pathological studies in zebrafish. The current protocol includes complete steps for the sgRNA design for the gene of interest, sgRNA in vitro transcription, Cas9 mRNA in vitro transcription, in vivo sgRNA screen for the one with the highest efficiency, donor plasmid design and construction, microinjection in zebrafish larvae, KI founder screen and zebrafish live imaging. Critical steps, troubleshooting tips, quality control methods, and advantages and applications of this protocol are included and discussed. This protocol assures quick and accurate results at a low cost and has been validated by multiple trials.

<meta charset="utf8"></head><body>荧光标记以可视化斑马鱼中的低表达蛋白质

Here, we present a protocol to label proteins with a fluorescence protein tag in zebrafish larvae, a newly developed and modified in vivo system especially useful for visualizing low-abundance proteins in zebrafish.

荧光标记以可视化斑马鱼中的低表达蛋白质

CRISPR/Cas9-mediated knock-in (KI) technology allows for easier fluorescent-protein tagging in zebrafish (Danio rerio), a preferred model organism for in ...

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 ...

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, ...

3dpf 斑马鱼幼虫的高通量 DNA 提取及基因分型研究

摘要

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此视频中的章节

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

使用斑马鱼作为体内模型系统分析 DNA 复制定时

分离和斑马鱼胚胎单细胞的表征

在发展中国家斑马鱼的前脑的神经祖细胞克隆相关的定时实时成像

筛选精子以快速分离斑马鱼的种系编辑

荧光标记以可视化斑马鱼中的低表达蛋白质

荧光标记以可视化斑马鱼中的低表达蛋白质

荧光标记以可视化斑马鱼中的低表达蛋白质

斑马鱼原位杂交染色的基因分型与定量

检查斑马鱼肌肉疾病模型中的肌肉再生