我们感谢女士出色的鱼牧保罗伊，史蒂夫Ekker pDB600质粒礼物。这些研究是由来自NHGRI批到SF。

1。选型和测试的保守序列的克隆<ol><li>首先必须找出潜在的调控序列进行功能测试。我们依赖于进化的序列保守性作为标准来选择序列，最经常使用的算法PhastCons，这是作为跟踪UCSC基因组浏览器（http://genome.ucsc.edu/cgi-bin/hgGateway）访问。然而，还有许多其他可用来评估跨物种的序列保守的算法。 </li><li>一旦我们选择了我们的序列，设计引物扩增基因组DNA的每个序列。应选择引物扩增保守的区域再加上30个或更多的碱基对任何一方。 </li><li>对于选定的序列的扩增，我们使用的宝LA TaqTM系统。其他聚合酶的工作，但重要的是要使用的混合，其中包括一个与校对能力的酶，以减少放大过程中引入的错误的机会。我们核实琼脂糖凝胶电泳扩增大小。 </li><li>我们使用的PCR 8/GW/TOPO TA克隆试剂盒（Invitrogen）的克隆到一个条目含有attL重组位点的载体的PCR产物，生成的网关条目克隆。克隆反应是新鲜的PCR产物更有效率，所以最好是一天的PCR克隆内执行。 DH5α菌株的转化是由奇霉素抗性选择。 </li><li>我们验证TA克隆的产物〜500ng的质粒用EcoRI消化释放插入，并确认的琼脂糖凝胶电泳插入的大小。我们建议测序验证插入的顺序和方向，也可用于测序引物扩增。 </li><li>从入门克隆的保守序列，非编码穿梭LR的重组，我们的网关的准备目标向量，pGW_cfosGFP1，使用网关LR Clonase II酶混合物（Invitrogen公司）。 DH5α菌株的转化是通过氨苄青霉素抗性选择。 </li><li>我们产品的LR重组核实消化〜500ng的质粒与EcoRV释放插入，并确认的琼脂糖凝胶电泳插入的大小。一旦已确定一个准确的克隆，我们准备了质粒DNA使用Qiagen公司HiSpeed​​ ®质粒MIDI套件。我们进一步纯化的质粒用QIAquick PCR纯化试剂盒，根据制造商的协议，并与30μLRNase的自由水洗脱的DNA。质粒是稀释浓度为125纳克/μL，并保存在4 ° C前注射。 </li><li>从pDB600 plasmid2 体外转录的RNA编码功能Tol2转座酶。我们净化使用Qiagen公司的Midi - PREP试剂盒的质粒，然后用XbaI线性10-20微克。 mRNA的合成进行了以下mMESSAGE mMACHINE包协议（Ambion公司）。 </li><li>我们净化和沉淀的RNA按试剂盒说明书。我们悬浮RNA的〜1μg/μl终浓度，或20μL一个反应，在核糖核酸酶自由水，紫外分光光度法和量化。我们还分析了〜琼脂糖凝胶电泳的RNA1μg验证全长转录。虽然标准的TAE或TBE凝胶是足够的，这种分析，转录试剂盒中包含的变性样品缓冲液应使用按试剂盒说明书。 </li><li>我们测试的功效，与已知的质粒（图1）执行注射每一批新的转座RNA。经核实，RNA分成小等分，并保存于-80 ° C，以避免反复解冻和重新冻结个人等份。 <img src="/files/ftp_upload/1722/1722fig1.jpg" alt="图1" /> 图1。斑马鱼胚胎注射用鼠标SOX10增强剂控制（上）明场（ 下 ）GFP荧光图像。每一批新的转座RNA是对疗效进行测试。 </li></ol> 2。斑马鱼的胚胎显微注射马赛克转基因分析<ol><li>我们从1.2毫米外径长丝在萨特的P - 97微管牵引器的毛细管玻璃拉显微注射针，程序设计，产生一个具有相当尖锐的锥形完整的chorions渗透强烈的提示。我们打​​破立体显微镜下手工技巧，外直径约15微米，使用干净的刀片和微米幻灯片。针，可一天前注射，并储存在有盖的控股菜要保持清洁。 </li><li>我们保持定期的光线暗的周期我们的斑马鱼，有14小时的光照，根据标准conditions3。前一天表演microinjections，我们成立了定时交配繁殖小坦克，每一个基槽组成，开槽插入，和一个塑料盖鱼。我们把三女两男每吨在并行行ANK。 </li><li>在上午的microinjections，光周期开始后不久，我们开始在干净的系统处理过的水相结合的鸡蛋生产的男性和女性。重要的是经常检查鱼，并奠定了几分钟内收集的鸡蛋，以及同步批次。蛋产量可维持两到三个小时，继续结合整个上午的新鲜的鱼群体。 </li><li>一个大口径，5 1 / 4“装有一个乳胶灯泡玻璃巴斯德吸管，我们排序为60 × 15毫米培养皿部分充满胚胎中2，在50个胚胎组，收集胚胎，并标记时间收集每道菜的盖子。 </li><li>一旦鱼已经开始铺设，我们准备了新鲜的注射液在离心管上冰混合以下： <table border="1"><tbody><tr><td>组件</td><td></td></tr><tr><td>转座子质粒股票（125ng/μl） </td><td>加入1μl </td></tr><tr><td>转座子RNA股票（175ng/μl） </td><td>加入1μl </td></tr><tr><td>酚红股（H 2 O 2％） </td><td> 0.5μL </td></tr><tr><td>核糖核酸酶自由水</td><td> 2.5μl </td></tr></tbody></table> 在控股菜奠定注射针头和充满吹打到广角端的是每个针注射液500 NL滴。后的液体通过毛细作用的提示，可以添加额外的解决方案共1.5-2μL。我们准备至少有两个针头每次注射液。 </li><li>充满针装入手持针气动的Pico -泵或类似的高压注射器，配置并连接到每个制造商的指示的氮气罐，人。我们通过测量微米幻灯片上驱逐到矿物油的液滴直径校准注入量。通常情况下，用20磅的压力的注射时间为120毫秒，将产生约1 NL液滴，但针直径的微小变化会影响这些参数之间的针头可能需要重新校准。 </li><li> 6 - 10X放大倍率的体视显微镜下进行注射。我们使用了一双细镊子举行的地方胚胎，推动通过蛋壳和稳定的压力到一个单元或两个细胞阶段的早期胚胎在蛋黄注射针头。理想的情况下，我们的地位在针尖蛋黄正下方的卵裂球。约1 NL注射液被驱逐和针撤回。对于每一个构造我们注入≥200胚胎。这也是非常重要的，每个收集胚胎离合器，一碟uninjected控制胚胎保存。 </li><li>注射完成后，我们通过清除未受精卵，胚胎受损，未能注射胚胎在卵裂球酚红没有排序胚胎。然后，我们在28.5 ° C，直到适当的时候观测孵化胚胎培养基中的胚胎。 </li></ol> 3。马赛克表达模式分析<ol><li>经过适当的培养时间，我们研究荧光注入胚胎。时间取决于内源性的斑马鱼基因的正常表达模式，但通常我们期待通过受精后第5天每天。 </li><li>我们研究epifluorescence装奥林巴斯MVX10 macroscope荧光的胚胎，但任何类似显微镜的低功耗成像能力的工作。如果有大量的胚胎死亡，或在第一天的异常发达，看看uninjected离合器的控制，看看他们看起来正常。如果控件的外观正常，最常见的问题是注入的DNA的成色不足。此外，在早期阶段的胚胎注入质粒总额敏感，所以它是可能的，是不准确的量化和太多的DNA注入。 </li><li>研究胚胎时，请记住，他们都将镶嵌注入的构造。仔细看所有的胚胎将有必要的，特别是如果预计域表达小。还记得的表达，可以动态;较强的荧光，第1天，第2天，胚胎负，第3天表达可能会显示第4天的东西可能会消失。 </li><li>我们选择一些更广泛的表达胚胎，并使用这些文件通过光学显微镜，荧光模式。经过5天的观察，胚胎，应返回到工厂的股票筹集的。在数月内，成年人可以进行筛选，生殖细胞传播的转基因。 </li></ol> 4。结果我们看到一个宽Variety模式和表达水平，取决于被分析的增强子元件。我们通常是在非常的组织特异性表达模式感兴趣，而且往往集中在骨架基因表达。往往集中在骨架基因表达。图2显示了马赛克表达模式与调节软骨和骨中的表达增强剂的例子，我们观察到，在我们注射的胚胎。最后，测试序列的很大一部分不规范组织的具体体现。对于这些，我们最常看到的非常小的荧光，也许数每胚胎细胞散。减的时候，我们可能会看到荧光，但只有在两种常见的部位为异位表达，表皮和骨骼肌肉（图3）。 <img src="/files/ftp_upload/1722/1722fig2.jpg" alt="图2" /> 图2。在注射胚胎中观察到的软骨和骨的表达模式的例子。 <img src="/files/ftp_upload/1722/1722fig3.jpg" alt="图3" /> 图3。有增强基因表达调控的相对位置之间的关联度不大。一个测试序列的很大一部分不规范的组织特异性表达。这些通常只有很少的荧光，或可能有两个常见的部位为异位表达：表皮和骨骼肌。 （上）明视图像（ 下 ）GFP的荧光图像。

<ol>
	<li>Fisher, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluating+the+biological+relevance+of+putative+enhancers+using+Tol2+transposon-mediated+transgenesis+in+zebrafish.">Evaluating the biological relevance of putative enhancers using Tol2 transposon-mediated transgenesis in zebrafish.</a> Nat Protoc. 1 (3), 1297-12 (2006).</li><li>Balciunas, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Harnessing+a+high+cargo-capacity+transposon+for+genetic+applications+in+vertebrates.">Harnessing a high cargo-capacity transposon for genetic applications in vertebrates.</a> PLoS Genet. 2 (11), e169-169 (2006).</li><li>Westerfield, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The Zebrafish Book. , (1995).</li></ol>

我们已经证明，在我们的实验室用于潜在的增强剂使用斑马鱼转基因的高效分析方法。大多数情况下，我们使用这种方法，寻找与人类基因相关的组织特异性的增强剂。尽管缺乏明显的序列同源性之间的人类和鱼类非编码序列，我们发现，这种方法通常是在成功地确定我们感兴趣的基因的增强剂。成功的关键因素，正在编制的质粒DNA和RNA的转座护理，注射和检查每个构造足够数量的胚胎。转基因?...

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		<div>
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		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
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<tr>
<td>Takara LA Taq</td>
<td>&nbsp;</td>
<td>Takara Bio Inc</td>
<td>RR02M</td>
<td>&nbsp;</td>
<tr>
<td>pCR8/GW/TOPO TA Cloning Kit</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>K2500-20</td>
<td>&nbsp;</td>
<tr>
<td>Gateway LR Clonase II enzyme Mix</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>11791-100</td>
<td>&nbsp;</td>
<tr>
<td>Library efficiency competent cells DH5&alpha;</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>12535-019</td>
<td>&nbsp;</td>
<tr>
<td>Library efficiency competent cells DB3.1</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>11782-018</td>
<td>&nbsp;</td>
<tr>
<td>mMESSAGE mMACHINE Kit</td>
<td>&nbsp;</td>
<td>Ambion</td>
<td>AM1340</td>
<td>&nbsp;</td>
<tr>
<td>HiSpeed Plasmid Midi Kit</td>
<td>&nbsp;</td>
<td>Qiagen</td>
<td>12643</td>
<td>&nbsp;</td>
<tr>
<td>QIAquick PCR Purification Kit </td>
<td>&nbsp;</td>
<td>Qiagen</td>
<td>28104</td>
<td>&nbsp;</td>
<tr>
<td>Flaming/Brown Micropipette Puller</td>
<td>&nbsp;</td>
<td>Sutter Instrument</td>
<td>P-97</td>
<td>&nbsp;</td>
<tr>
<td>Pneumatic PicoPump</td>
<td>&nbsp;</td>
<td>World Precision Instruments</td>
<td>SYS-PV820</td>
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mosaic zebrafish transgenesis for evaluating enhancer sequences

人类基因组序列的完成，以及其他许多物种，突出了把特定功能的非编码序列的挑战。由非编码的基因组的一部分进行的一个突出功能是调节基因的转录;但是，有没有有效的方法来大致预测的主要DNA序列的顺式调控元件。我们已经开发出一种高效的协议功能评估潜在的顺式调控，通过斑马鱼转基因的元素。我们的方法提供了发育的重要基因的细胞培养为基础的技术，显着的优势，因为它提供信息的空间和时间的基因调控。相反，它是更快和更昂贵的比类似的实验，在转基因小鼠，我们经常将其应用到人类基因组序列。在这里，我们证明我们的方法来测试序列克隆序列的养护和我们的协议的基础上，显微注射到斑马鱼的胚胎中选择元素。

Watch this Scientific Journal Video about Mosaic Zebrafish Transgenesis for Evaluating Enhancer Sequences at JoVE.com

Mosaic Zebrafish Transgenesis for Evaluating Enhancer Sequences

我们证明我们的方法来寻找潜在发育调控基因的增强子元件和评估它们的功能，通过镶嵌的斑马鱼转基因。

马赛克斑马鱼转基因增强子序列，以评估

The completion of the human genome sequence, along with that of many other species, has highlighted the challenge of ascribing specific function to non ...

mosaic-zebrafish-transgenesis-for-evaluating-enhancer-sequences

Research

JoVE Journal

Biology

13.9K 次观看.  University of Pennsylvania. 我们证明我们的方法来寻找潜在发育调控基因的增强子元件和评估它们的功能，通过镶嵌的斑马鱼转基因。

视频: Mosaic Zebrafish Transgenesis for Evaluating Enhancer Sequences

Zebrafish Brain Ventricle Injection

Proper brain ventricle formation during embryonic brain development is required for normal brain function.  Brain ventricles are the highly conserved cavities within the brain that are filled with cerebrospinal fluid.  In zebrafish, after neural tube formation, the neuroepithelium undergoes a series of constrictions and folds while it fills with fluid resulting in brain ventricle formation.  In order to understand the process of ventricle formation, and the neuroepithelial shape changes that occur at the same time, we needed a way to visualize the ventricle space in comparison to the brain tissue.  However, the nature of transparent zebrafish embryos makes it difficult to differentiate the tissue from the ventricle space.  Therefore, we developed a brain ventricle injection technique where the ventricle space is filled with a fluorescent dye and imaged by brightfield and fluorescent microscopy.  The brightfield and the fluorescent images are then processed and superimposed in Photoshop.  This technique allows for visualization of the ventricle space with the fluorescent dye, in comparison to the shape of the neuroepithelium in the brightfield image.  Brain ventricle injection in zebrafish can be employed from 18 hours post fertilization through early larval stages.  We have used this technique extensively in our studies of brain ventricle formation and morphogenesis as well as in characterizing brain morphogenesis mutants (1-3).

After neural tube formation, the neuroepithelium constricts and folds while the tube fills with embryonic cerebrospinal fluid (eCSF) to form the embryonic brain ventricles. We developed this ventricle injection technique to better visualize the fluid filled space in contrast to the neuroepithelial shape in a live embryo.

斑马鱼脑室注射液

Proper brain ventricle formation during embryonic brain development is required for normal brain function.  Brain ventricles are the highly conserved ...

科研

生物学

RNAi Mediated Gene Knockdown and Transgenesis by Microinjection in the Necromenic Nematode Pristionchus pacificus

Although it is increasingly affordable for emerging model organisms to obtain completely sequenced genomes, further in-depth gene function and expression analyses by RNA interference and stable transgenesis remain limited in many species due to the particular anatomy and molecular cellular biology of the organism. For example, outside of the crown group Caenorhabditis that includes Caenorhabditis elegans3, stably transmitted transgenic lines in non-Caenorhabditis species have not been reported in this specious phylum (Nematoda), with the exception of Strongyloides stercoralis4 and Pristionchus pacificus5. To facilitate the expanding role of P. pacificus in the study of development, evolution, and behavior6-7, we describe here the current methods to use microinjection for making transgenic animals and gene knock down by RNAi. Like the gonads of C. elegans and most other nematodes, the gonads of P. pacificus is syncitial and capable of incorporating DNA and RNA into the oocytes when delivered by direct microinjection. Unlike C. elegans however, stable transgene inheritance and somatic expression in P. pacificus requires the addition of self genomic DNA digested with endonucleases complementary to the ends of target transgenes and coinjection markers5. The addition of carrier genomic DNA is similar to the requirement for transgene expression in Strongyloides stercoralis4 and in the germ cells of C. elegans. However, it is not clear if the specific requirement for the animals' own genomic DNA is because P. pacificus soma is very efficient at silencing non-complex multi-copy genes or that extrachromosomal arrays in P. pacificus require genomic sequences for proper kinetochore assembly during mitosis. The ventral migration of the two-armed (didelphic) gonads in hermaphrodites further complicates the ability to inject both gonads in individual worms8. We also demonstrate the use of microinjection to knockdown a dominant mutant (roller,tu92) by injecting double-stranded RNA (dsRNA) into the gonads to obtain non-rolling F1 progeny. Unlike C. elegans, but like most other nematodes, P. pacificus PS312 is not receptive to systemic RNAi via feeding and soaking and therefore dsRNA must be administered by microinjection into the syncitial gonads. In this current study, we hope to describe the microinjection process needed to transform a Ppa-egl-4 promoter::GFP fusion reporter and knockdown a dominant roller prl-1 (tu92) mutant in a visually informative protocol.

RNAi Mediated Gene Knockdown and Transgenesis by Microinjection in the Necromenic Nematode Pristionchus pacificus

In model organisms, transgenesis can manipulate gene functions while RNAi can knockdown specific mRNA transcripts 1-2. This protocol aims to illustrate the techniques needed to introduce stably transmitted DNA and transient double stranded RNA into the necromenic nematode Pristionchus pacificus for studies in evolutionary, developmental, and behavioral biology.

通过显微注射介导的RNAi Necromenic线虫基因敲除和转基因 Pristionchus pacificus</em

Although it is increasingly affordable for emerging model organisms to obtain completely sequenced genomes, further in-depth gene function and expression ...

In vivo Electroporation of Morpholinos into the Adult Zebrafish Retina

Many devastating inherited eye diseases result in progressive and irreversible blindness because humans cannot regenerate dying or diseased retinal neurons. In contrast, the adult zebrafish retina possesses the robust ability to spontaneously regenerate any neuronal class that is lost in a variety of different retinal damage models, including retinal puncture, chemical ablation, concentrated high temperature, and intense light treatment 1-8. Our lab extensively characterized regeneration of photoreceptors following constant intense light treatment and inner retinal neurons after intravitreal ouabain injection 2, 5, 9. In all cases, resident M&uuml;ller glia re-enter the cell cycle to produce neuronal progenitors, which continue to proliferate and migrate to the proper retinal layer, where they differentiate into the deficient neurons. 
	We characterized five different stages during regeneration of the light-damaged retina that were highlighted by specific cellular responses. We identified several differentially expressed genes at each stage of retinal regeneration by mRNA microarray analysis 10. Many of these genes are also critical for ocular development. To test the role of each candidate gene/protein during retinal regeneration, we needed to develop a method to conditionally limit the expression of a candidate protein only at times during regeneration of the adult retina. 
	Morpholino oligos are widely used to study loss of function of specific proteins during the development of zebrafish, Xenopus, chick, mouse, and tumors in human xenografts 11-14. These modified oligos basepair with complementary RNA sequence to either block the splicing or translation of the target RNA. Morpholinos are stable in the cell and can eliminate or "knockdown" protein expression for three to five days 12. 
	Here, we describe a method to efficiently knockdown target protein expression in the adult zebrafish retina. This method employs lissamine-tagged antisense morpholinos that are injected into the vitreous of the adult zebrafish eye. Using electrode forceps, the morpholino is then electroporated into all the cell types of the dorsal and central retina. Lissamine provides the charge on the morpholino for electroporation and can be visualized to assess the presence of the morpholino in the retinal cells. 
	Conditional knockdown in the retina can be used to examine the role of specific proteins at different times during regeneration. Additionally, this approach can be used to study the role of specific proteins in the undamaged retina, in such processes as visual transduction and visual processing in second order neurons.

In vivo Electroporation of Morpholinos into the Adult Zebrafish Retina

A method to conditionally knockdown a target protein&#x2019;s expression in the adult zebrafish retina is described, which involves intravitreally injecting antisense morpholinos and electroporating them into the retina. The resulting protein is knocked down for several days, which allows testing the protein&#x2019;s role in the regenerating or intact retina.

在体内电穿孔Morpholinos到成年斑马鱼视网膜

Many devastating inherited eye diseases result in progressive and irreversible blindness because humans cannot regenerate dying or diseased retinal ...

In vivo Electroporation of Morpholinos into the Regenerating Adult Zebrafish Tail Fin

Certain species of urodeles and teleost fish can regenerate their tissues. Zebrafish have become a widely used model to study the spontaneous regeneration of adult tissues, such as the heart1, retina2, spinal cord3, optic nerve4, sensory hair cells5, and fins6.
The zebrafish fin is a relatively simple appendage that is easily manipulated to study multiple stages in epimorphic regeneration. Classically, fin regeneration was characterized by three distinct stages: wound healing, blastema formation, and fin outgrowth. After amputating part of the fin, the surrounding epithelium proliferates and migrates over the wound. At 33 &deg;C, this process occurs within six hours post-amputation (hpa, Figure 1B)6,7. Next, underlying cells from different lineages (ex. bone, blood, glia, fibroblast) re-enter the cell cycle to form a proliferative blastema, while the overlying epidermis continues to proliferate (Figure 1D)8. Outgrowth occurs as cells proximal to the blastema re-differentiate into their respective lineages to form new tissue (Figure 1E)8. Depending on the level of the amputation, full regeneration is completed in a week to a month.
The expression of a large number of gene families, including wnt, hox, fgf, msx, retinoic acid, shh, notch, bmp, and activin-betaA genes, is up-regulated during specific stages of fin regeneration9-16. However, the roles of these genes and their encoded proteins during regeneration have been difficult to assess, unless a specific inhibitor for the protein exists13, a temperature-sensitive mutant exists or a transgenic animal (either overexpressing the wild-type protein or a dominant-negative protein) was generated7,12. We developed a reverse genetic technique to quickly and easily test the function of any gene during fin regeneration.
Morpholino oligonucleotides are widely used to study loss of specific proteins during zebrafish, Xenopus, chick, and mouse development17-19. Morpholinos basepair with a complementary RNA sequence to either block pre-mRNA splicing or mRNA translation. We describe a method to efficiently introduce fluorescein-tagged antisense morpholinos into regenerating zebrafish fins to knockdown expression of the target protein. The morpholino is micro-injected into each blastema of the regenerating zebrafish tail fin and electroporated into the surrounding cells. Fluorescein provides the charge to electroporate the morpholino and to visualize the morpholino in the fin tissue.
This protocol permits conditional protein knockdown to examine the role of specific proteins during regenerative fin outgrowth. In the Discussion, we describe how this approach can be adapted to study the role of specific proteins during wound healing or blastema formation, as well as a potential marker of cell migration during blastema formation.

In vivo Electroporation of Morpholinos into the Regenerating Adult Zebrafish Tail Fin

We describe a method to conditionally knockdown the expression of a target protein during adult zebrafish fin regeneration. This technique involves micro-injecting and electroporating antisense oligonucleotide morpholinos into fin tissue, which allows testing the protein&#x2019;s role in various stages of fin regeneration, including wound healing, blastema formation, and regenerative outgrowth.

再生的成年斑马鱼尾鳍在吗啉代入体内电穿孔

Certain species of urodeles and teleost fish can regenerate their tissues. Zebrafish have become a widely used model to study the spontaneous regeneration ...

Laser-inflicted Injury of Zebrafish Embryonic Skeletal Muscle

Various experimental approaches have been used in mouse to induce muscle injury with the aim to study muscle regeneration, including myotoxin injections (bupivacaine, cardiotoxin or notexin), muscle transplantations (denervation-devascularization induced regeneration), intensive exercise, but also murine muscular dystrophy models such as the mdx mouse (for a review of these approaches see 1). In zebrafish, genetic approaches include mutants that exhibit muscular dystrophy phenotypes (such as runzel2 or sapje3) and antisense oligonucleotide morpholinos that block the expression of dystrophy-associated genes4. Besides, chemical approaches are also possible, e.g. with Galanthamine, a chemical compound inhibiting acetylcholinesterase, thereby resulting in hypercontraction, which eventually leads to muscular dystrophy5. However, genetic and pharmacological approaches generally affect all muscles within an individual, whereas the extent of physically inflicted injuries are more easily controlled spatially and temporally1. Localized physical injury allows the assessment of contralateral muscle as an internal control. Indeed, we recently used laser-mediated cell ablation to study skeletal muscle regeneration in the zebrafish embryo6, while another group recently reported the use of a two-photon laser (822 nm) to damage very locally the plasma membrane of individual embryonic zebrafish muscle cells7.
Here, we report a method for using the micropoint laser (Andor Technology) for skeletal muscle cell injury in the zebrafish embryo. The micropoint laser is a high energy laser which is suitable for targeted cell ablation at a wavelength of 435 nm. The laser is connected to a microscope (in our setup, an optical microscope from Zeiss) in such a way that the microscope can be used at the same time for focusing the laser light onto the sample and for visualizing the effects of the wounding (brightfield or fluorescence). The parameters for controlling laser pulses include wavelength, intensity, and number of pulses.
Due to its transparency and external embryonic development, the zebrafish embryo is highly amenable for both laser-induced injury and for studying the subsequent recovery. Between 1 and 2 days post-fertilization, somitic skeletal muscle cells progressively undergo maturation from anterior to posterior due to the progression of somitogenesis from the trunk to the tail8, 9. At these stages, embryos spontaneously twitch and initiate swimming. The zebrafish has recently been recognized as an important vertebrate model organism for the study of tissue regeneration, as many types of tissues (cardiac, neuronal, vascular etc.) can be regenerated after injury in the adult zebrafish10, 11.

The method presented here comprises the precise injury of live zebrafish embryos with high-energy laser pulses and the subsequent analysis of these injuries and their recovery with time. We also show how genetically labeled single or groups of skeletal muscle cells can be tracked during and after laser light induced damage.

激光伤害的斑马鱼胚胎骨骼肌

Various experimental approaches have been used in mouse to induce muscle injury with the aim to study muscle regeneration, including myotoxin injections ...

The Tomato/GFP-FLP/FRT Method for Live Imaging of Mosaic Adult Drosophila Photoreceptor Cells

The Drosophila eye is widely used as a model for studies of development and neuronal degeneration. With the powerful mitotic recombination technique, elegant genetic screens based on clonal analysis have led to the identification of signaling pathways involved in eye development and photoreceptor (PR) differentiation at larval stages. We describe here the Tomato/GFP-FLP/FRT method, which can be used for rapid clonal analysis in the eye of living adult Drosophila. Fluorescent photoreceptor cells are imaged with the cornea neutralization technique, on retinas with mosaic clones generated by flipase-mediated recombination. This method has several major advantages over classical histological sectioning of the retina: it can be used for high-throughput screening and has proved an effective method for identifying the factors regulating PR survival and function. It can be used for kinetic analyses of PR degeneration in the same living animal over several weeks, to demonstrate the requirement for specific genes for PR survival or function in the adult fly. This method is also useful for addressing cell autonomy issues in developmental mutants, such as those in which the establishment of planar cell polarity is affected.

The Tomato/GFP-FLP/FRT Method for Live Imaging of Mosaic Adult Drosophila Photoreceptor Cells

The Tomato/GFP-FLP/FRT method involves visualizing mosaic photoreceptor cells in living Drosophila. It can be used to follow individual photoreceptor cell fates in the retina for days or weeks. This method is ideal for studies of retinal degeneration and neurodegenerative diseases or photoreceptor cell development.

番茄/ GFP-FLP / FRT方法马赛克成人的实时成像<em&gt;果蝇</em&gt;感光细胞

The Drosophila eye is widely used as a model  for studies of development and neuronal degeneration. With the powerful mitotic  recombination technique, ...

Separation of Spermatogenic Cell Types Using STA-PUT Velocity Sedimentation

Mammalian spermatogenesis is a complex differentiation process that occurs in several stages in the seminiferous tubules of the testes. Currently, there is no reliable cell culture system allowing for spermatogenic differentiation in vitro, and most biological studies of spermatogenic cells require tissue harvest from animal models like the mouse and rat. Because the testis contains numerous cell types - both non-spermatogenic (Leydig, Sertoli, myeloid, and epithelial cells) and spermatogenic (spermatogonia, spermatocytes, round spermatids, condensing spermatids and spermatozoa) - studies of the biological mechanisms involved in spermatogenesis require the isolation and enrichment of these different cell types. The STA-PUT method allows for the separation of a heterogeneous population of cells - in this case, from the testes - through a linear BSA gradient. Individual cell types sediment with different sedimentation velocity according to cell size, and fractions enriched for different cell types can be collected and utilized in further analyses. While the STA-PUT method does not result in highly pure fractions of cell types, e.g. as can be obtained with certain cell sorting methods, it does provide a much higher yield of total cells in each fraction (~1 x 108 cells/spermatogenic cell type from a starting population of 7-8 x 108 cells). This high yield method requires only specialized glassware and can be performed in any cold room or large refrigerator, making it an ideal method for labs that have limited access to specialized equipment like a fluorescence activated cell sorter (FACS) or elutriator.

The STA-PUT method allows for the separation of different populations of spermatogenic cells based on size and density.

生精细胞类型使用STA把速度沉降分离

Mammalian spermatogenesis is a complex  differentiation process that occurs in several stages in the seminiferous  tubules of the testes. Currently, there ...

Automated Analysis of Dynamic Ca2+ Signals in Image Sequences

Intracellular Ca2+ signals are commonly studied with fluorescent Ca2+ indicator dyes and microscopy techniques. However, quantitative analysis of Ca2+ imaging data is time consuming and subject to bias. Automated signal analysis algorithms based on region of interest (ROI) detection have been implemented for one-dimensional line scan measurements, but there is no current algorithm which integrates optimized identification and analysis of ROIs in two-dimensional image sequences. Here an algorithm for rapid acquisition and analysis of ROIs in image sequences is described. It utilizes ellipses fit to noise filtered signals in order to determine optimal ROI placement, and computes Ca2+ signal parameters of amplitude, duration and spatial spread. This algorithm was implemented as a freely available plugin for ImageJ (NIH) software. Together with analysis scripts written for the open source statistical processing software R, this approach provides a high-capacity pipeline for performing quick statistical analysis of experimental output. The authors suggest that use of this analysis protocol will lead to a more complete and unbiased characterization of physiologic Ca2+ signaling.

Automated Analysis of Dynamic Ca2+ Signals in Image Sequences

Here a novel region of interest analysis protocol based on sorting best-fit ellipses assigned to regions of positive signal within two-dimensional time lapse image sequences is demonstrated. This algorithm may enable investigators to comprehensively analyze physiological Ca2+ signals with minimal user input and bias.

动态钙的自动分析<sup&gt; 2 +</sup&gt;在图像序列信号

Intracellular Ca2+ signals are commonly studied with fluorescent Ca2+ indicator dyes and microscopy techniques. However, quantitative analysis of Ca2+ ...

A Simplified System for Evaluating Cell Mechanosensing and Durotaxis In Vitro

The composition and mechanical properties of the extracellular matrix are highly variable between tissue types. This connective tissue stroma diversity greatly impacts cell behavior to regulate normal and pathologic processes including cell proliferation, differentiation, adhesion signaling and directional migration. In this regard, the innate ability of certain cell types to migrate towards a stiffer, or less compliant matrix substrate is referred to as durotaxis. This phenomenon plays an important role during embryonic development, wound repair and cancer cell invasion. Here, we describe a straightforward assay to study durotaxis, in vitro, using polydimethylsiloxane (PDMS) substrates. Preparation of the described durotaxis chambers creates a rigidity interface between the relatively soft PDMS gel and a rigid glass coverslip. In the example provided, we have used these durotaxis chambers to demonstrate a role for the cdc42/Rac1 GTPase activating protein, cdGAP, in mechanosensing and durotaxis regulation in human U2OS osteosarcoma cells. This assay is readily adaptable to other cell types and/or knockdown of other proteins of interests to explore their respective roles in mechanosignaling and durotaxis.

A Simplified System for Evaluating Cell Mechanosensing and Durotaxis In Vitro

Many mammalian cells preferentially migrate towards a more rigid matrix or substrate through durotaxis. The goal of this protocol is to provide a simple in vitro system that can be used to study and manipulate cell durotaxis behaviors by incorporating polydimethylsiloxane (PDMS) substrates of defined rigidity, interfacing with glass coverslips.

为评估细胞Mechanosensing和Durotaxis简化系统<em&gt;体外</em

The composition and mechanical properties of the extracellular matrix are highly variable between tissue types. This connective tissue stroma diversity ...

Nephrotoxin Microinjection in Zebrafish to Model Acute Kidney Injury

The kidneys are susceptible to harm from exposure to chemicals they filter from the bloodstream. This can lead to organ injury associated with a rapid decline in renal function and development of the clinical syndrome known as acute kidney injury (AKI). Pharmacological agents used to treat medical circumstances ranging from bacterial infection to cancer, when administered individually or in combination with other drugs, can initiate AKI. Zebrafish are a useful animal model to study the chemical effects on renal function in vivo, as they form an embryonic kidney comprised of nephron functional units that are conserved with higher vertebrates, including humans. Further, zebrafish can be utilized to perform genetic and chemical screens, which provide opportunities to elucidate the cellular and molecular facets of AKI and develop therapeutic strategies such as the identification of nephroprotective molecules. Here, we demonstrate how microinjection into the zebrafish embryo can be utilized as a paradigm for nephrotoxin studies.

Renal injuries incurred from nephrotoxins, which include drugs ranging from antibiotics to chemotherapeutics, can result in complex disorders whose pathogenesis remains incompletely understood. This protocol demonstrates how zebrafish can be used for disease modeling of these conditions, which can be applied to the identification of renoprotective measures.

肾毒素显微注射斑马鱼到模型急性肾损伤

The kidneys are susceptible to harm from exposure to chemicals they filter from the bloodstream. This can lead to organ injury associated with a rapid ...