这项工作是通过美国国立卫生研究院R15区授予约翰霍恩（镍氢电池; R15MH083221）资助。

1。转基因胚胎<ol><li>这里我们使用了斑马鱼转基因株系，通过转座子介导的增强陷阱的方法8。转基因插入包括编码序列KalTA4，一个优化版本的GAL4转录激活斑马鱼，和红色荧光蛋白，mCherry。增强陷阱的方法产量线的转基因斑马鱼，这种转基因盒表达内源性增强子序列的控制之下。因此，根据特定的增强子序列的身份，KalTA4和mCherry表达是本地化到发育中的大脑的特定区域。 ET（zic4:Gal4TA4，无人机:mCherry）hzm5线，我们在这里使用，表达本地化的几个地点，脑，后脑和具有特别重要的意义，在这里，嗅球8（见图1。） 。鉴于这种表达方式，和本地化的基因组内的录像带，转基因预测zic4 发起人 8的控制之下（ <a href="http://www.helmholtz-muenchen.de/en/idg/groups/neuroimaging/lines_distel/main">见</a>也http://www.helmholtz-muenchen.de/en/idg/groups/ <a href="http://www.helmholtz-muenchen.de/en/idg/groups/neuroimaging/lines_distel/main">影像学/ lines_distel /主</a> ）。 </li><li>成人ET（zic4:Gal4TA4，无人机:mCherry）hzm5鱼杂合子转基因插入通常与WT（AB）鱼交配，导致约50％的转基因后代。 （对于转基因后代的比例更高，两个杂合子可以交配。） </li><li>为了阻止色素形成和促进荧光成像，100μM的N -苯基硫脲嘧啶（PTU）在6-8 HPF开始对待转基因胚胎。 </li></ol> 2。安装胚胎<ol><li> 24-28 HPF，转移胚胎蛋水，无机动部队。确定使用荧光显微镜的表达mCherry的转基因胚胎。取出用细镊子的转基因胚胎绒毛膜膜。传输中解脱出来，从胚胎绒毛膜培养皿中含有电加0.02％的缓冲tricaine [电穿孔缓冲:180毫米氯化钠，氯化钾5毫米，1.8毫米氯化钙 2，5毫米的HEPES，pH值7.2] 4 。允许2分钟的麻醉效果，然后再继续安装步骤。 </li><li>微波解决方案，然后放置到一个34-37 ° C培养箱解决方案，准备一个0.5％的低融点琼脂糖电缓冲区加上tricaine解决方案。一旦琼脂糖溶液的温度平衡，将大幅下降（约500μL）的琼脂糖溶液60毫米培养皿的中心。 6-8麻醉胚胎的琼脂糖下降。 </li><li>使用镊子（或减少microloader吸管提示罚款结束）的罚款，位置的胚胎，它们都面临着同一方向，并在一个垂直的行对齐。巩固琼脂糖和陷阱的胚胎中的位置，放到一个大培养皿中，用2-3毫米的冰盘。 </li><li>一旦琼脂糖凝固后，洪水电缓冲区加上tricaine菜，以确保该解决方案完全覆盖凝固的琼脂糖下降。 </li></ol> 3。显微注射DNA的表达质粒<ol><li>准备使用的MIDI或Maxi准备质粒提取试剂盒（如Qiagen公司）的绿色荧光蛋白表达质粒。挂起到终浓度为0.5μg/μL无菌水加0.03％酚红质粒。对于嗅球针对实验在这里，我们使用多个Gal4的结合位点（14无人机串联序列和基底鱼发起人，E1B）控制下的EGFP编码序列的表达质粒。将使用这种质粒，transgenically表达的KalTA4转录因子的细胞能够表达绿色荧光蛋白，因此，调解有针对性的表达绿色荧光蛋白。 注:在这里，我们使用的DNA注射，应针对发育中的大脑的其他地区适用的可视化酚红，只要该地区可以从心室​​访问。针对其他类型的细胞，需要荧光可视化，肾上腺素或Quantam点染料可能被用于可视化荧光灯照明下注射。 </li><li>显微注射移液器可以采用水平或垂直的吸管拉马。热拉强度设置会有所不同拉马，加热元件的类型和玻璃毛细管的类型。 使用一个萨特的P - 30垂直拉拔器，使用了980的热定型和拉力为960，生产薄壁硼硅毛细管玻璃细丝长，逐渐变细，尖锐的移液器[萨特仪器＃BF100 - 78 - 10]。这些设置拉Microinjetion移液器是密封的，注射前必须打破。 </li><li>使用microloader枪头，加1-2微升DNA质粒溶液注射针。 </li><li>山和争取到的高压喷射系统移液器支架装入吸管。 </li><li>回破用细镊子装入吸管，直到压力脉冲产生膨化释放红色的DNA溶液。另外，小技巧，可以迅速穿透水下，折叠实验室Kimwipe被打破。 注:因为注射移液器最初密封，必须手动回破，针尖大小和注射量是可变的。理想的注射移液器需要3-5注射脉冲完全填补24 HPF胚胎（100毫秒脉冲注入40 PSI）的前脑脑室发展。 </li><li>质粒DNA溶液注入到发育中的大脑脑室这种DNA是迫使相邻，和闰成，注定要形成嗅球的大脑区域。嗅球是来自细胞在脑脑室前壁。因此，通过插入脑室注射针等移液器指向是对发育中的大脑前结束，高压喷射力量的DNA和相邻的准嗅球。注入的DNA，直到红色染料是清晰可见脑脑室前结束。因为DNA立即开始弥漫，稀释的DNA，重要的是进行电步骤尽快注射后（例如，10秒内）。 </li></ol> 4。电穿孔<ol><li>使用自建草铂（草技术＃E2）皮下电极的电极进行电穿孔。电极连接到一个手持式探头，并最终铂电极之间的距离应为1毫米（在建筑的细节见 9） 。方波电脉冲产生的电极连接到一个草SD - 9刺激器（草技术，型号SD - 9）。 </li><li>设置SD - 9刺激产生70伏单，5毫秒电脉冲。放置电极，正电极定位毗邻的胚胎前结束，而负电极后的胚胎头。手动启动〜7-8使用的SD - 9刺激的单一模式开关的电脉冲，每个脉冲分离〜1秒。适当的电压将取决于对胚胎的年龄和所使用的电压源。年轻的胚胎需要较低的电压，以避免胚胎的损害，而年纪大的胚胎需要更高的电压 ，启动电7。 </li><li>允许胚胎，至少10分钟后，电恢复之前，使他们从琼脂糖。 </li><li>用细镊子，免费琼脂糖胚胎轻轻跟踪胚胎的轮廓，同时避免与胚胎本身的实际接触。 </li><li>一旦释放，返回的胚胎蛋水（与机动部队如果需要的话）。保持在28 ° C，直到他们将GFP表达成像。 </li></ol> 5。成像安装胚胎<ol><li>胚胎转移，从鸡蛋的水，加上警察机动部队蛋水加0.02％tricaine。允许几分钟的麻醉效果，然后再继续安装步骤。 </li><li>微波解决方案，然后放置到一个34-37 ° C培养箱解决方案，准备一个0.5％的低融点琼脂糖鸡蛋水加tricaine解决方案。一旦琼脂糖溶液的温度平衡，将大幅下降（约300μL）的琼脂糖溶液到35毫米的培养皿的中心。添加一个麻醉胚胎的琼脂糖下降。 </li><li>使用镊子（或减少microloader吸管提示罚款结束）的罚款，位置的胚胎，他们是正直的，使它能够可视化的​​背视图，发育中的大脑与一个堂堂正正的显微镜（倒置的范围将需要不同的胚胎几何和不同的菜，可以从下面的可视化 ;看到12）。琼脂糖巩固放置到一个大培养皿中，用2-3毫米的冰菜。用细镊子将有可能在凝固过程中需要重新定位，以确保胚胎不落在其副作用。 </li><li>一旦琼脂糖凝固，洪水盘鸡蛋水加tricaine确保该解决方案完全覆盖凝固的琼脂糖下降。 </li></ol> 6。代表性的成果: 虽然GFP的表达，可以观察到早6个小时后，电，在这里，我们显示了从胚胎电后第4天，充分发展的目标细胞的突起结构图像。的ET（zic4:Gal4TA4，无人机:mCherry）hzm5转基因增强子陷阱线显示mCherry组成性表达（和KalTA4）在后脑，小脑，前脑，胚胎嗅球，在5天受精后（图1A和1C）。胚胎有一个UAS - GFP表达质粒针对性电（如上所述），显示绿色荧光蛋白的表达，是限制发展嗅球细胞（图1B，1D及1E）注册成立。只有细胞transgenically表达的KalTA4转录因子能够激活这个UAS - GFP的质粒表达。这个团到非转基因胚胎的质粒不会导致任何可察觉的GFP表达。因此，它是有针对性的UAS - GFP和本地化的KalTA4驱动程序，导致发展的嗅球细胞中GFP的独家表达的转基因表达电的联合行动。表达GFP的细胞显示出典型的轴突预测嗅球僧帽细胞（图1B和1D）10。为了形象化轴突的预测，在图1B和1D的图像被暴露在胞体的区域过度曝光高层次。一个暴露的较低水平（图1E）显示，表达GFP的细胞体确实本地化嗅球（比较图1C及1E;灰线，标志着嗅球边境）。 <img src="/files/ftp_upload/2964/2964fig1.jpg" alt="图1"/> 图1。针对性的5 DPF的斑马鱼胚胎的嗅球GFP的表达。 ET（zic4:Gal4TA4，无人机:mCherry）hzm5转基因增强子陷阱线，显示构mCherry表达在后脑，小脑，前脑和嗅球（A和 C，mCherry荧光显示为红色）。这mCherry表达介导的转基因Gal4的表达。胚胎已与UAS - GFP的表达载体，电穿孔28 HPF，显示绿色荧光蛋白的表达有针对性的在受精后5天的嗅球（B，D，和 E，绿色荧光绿色显示相同的胚胎，并在A C）。一个曝光的图片的底部（e）显示，GFP的表达是有限的嗅球细胞机构。灰线表示嗅球的边界。明场（灰度），绿色荧光（绿色），和红色荧光（红色）图像都是用Olympus BX60荧光显微镜。

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没有利益冲突的声明。

在这里，我们描述了在斑马鱼体内电穿孔法利用增强剂陷阱Gal4的转基因株系的目标电穿孔转基因表达的细胞特定的人口在发展中国家的嗅球。这种方法结合在体内电穿孔7增强剂陷阱转基因株系8介导的细胞类型的具体表达了良好的时间分辨率。虽然我们在这里所描述的嗅球的目标， 在体内电穿孔可用于其他地区的神经系统的发育2-7目标，并增强子陷阱线与Gal4的针对性体现在许多不同的特定细胞类型或组织8，11。电穿孔技术，当然，这也应该适合其他combinatioral LexA或春节系统13，14，15，如遗传方式。电穿孔的一个主要优点是，有没有需要给出一个合适的Gal4的行提供的额外的转基因株系。这样可以节省6个月内。 允许在任何神经系统发展的特定阶段的兴趣 7成神经细胞及其前体掺入的寡核苷酸在体内电。时间分辨率是亏损的功能分析特别有利，因为它可以规避与目标基因在早期发育阶段的基本职能的问题。我们在这里描述的方法也可以用来纳入丧失功能的试剂，包括RNA干扰寡核苷酸或构造和吗啉反义寡核苷酸4，7 。如显性负蛋白表达质粒或短发夹RNA干扰质粒质粒驱动的试剂也可以放在一个GAL4 UAS的控制下，允许精确的细胞类型特异性表达GFP的表达，我们已经表明这里。

试剂: <ul><li>琼脂糖，低融点（Sigma公司，A9414） </li><li>质粒的MIDI或Maxi制备的试剂盒（如Qiagen公司，＃12643] </li><li>酚红（Sigma公司，P3532） </li></ul>设备: <ul><li>毛细管玻璃长丝; ID = 0.78毫米，外径为1.0毫米（萨特仪器，＃BF100 - 78 - 10） </li><li>草SD - 9刺激（草技术，型号SD - 9） </li><li>草铂皮下电极，直针（草技术，＃E2） </li><li>微型装载机枪头（Eppendorf公司，＃X22703R） </li><li>移液器拉马（如萨特，模型P - 30） </li><li>移液器拉马（如萨特，模型P - 30） </li></ul>

targeting olfactory bulb neurons using combined in vivo electroporation and gal4-based enhancer trap zebrafish lines

在体内电穿孔是一种提供质粒DNA的表达，RNAi试剂，和吗啉反义寡核苷酸对胚胎发育的特定区域， 包括 C语言的强大方法线虫 ，鸡， 爪蟾 ，斑马鱼和小鼠1。在斑马鱼体内电穿孔已被证明有良好的空间和时间分辨率为提供这些试剂 2-7 。这种方法的时间分辨率是重要的，因为它允许这些试剂纳入发展的特定阶段。此外，因为电穿孔载体的表达发生在6小时内 7比转基因的方法，这种方法更及时。虽然空间分辨率可以非常精确的针对单个细胞2，6时，往往是最好将其纳入一个特定的细胞群，在一个组织或结构的试剂。当针对多个细胞， 在体内电穿孔是有效的传递到一个特定区域的胚胎;然而，尤其是在神经系统发育，它是很难的目标，仅仅通过特定的细胞类型空间上离散的电。另外，增强陷阱的转基因株系提供优良的转基因 8细胞类型的具体体现。在这里，我们描述了一个方法，结合体内电穿孔7，9 GAL4基于转基因的增强子陷阱线与空间上离散的专门针对发展中国家的斑马鱼嗅球的神经元。 ET（zic4:Gal4TA4，无人机:mCherry）hzm5（原GA80_9）增强子陷阱线前面所述8，显示斑马鱼的目标mCherry介导的转基因表达在发育中的大脑的多个区域，包括后脑优化Gal4的（KalTA4）转录激活因子，小脑，前脑和嗅球。要明确目标GFP表达到嗅球，与绿色荧光蛋白基因的编码序列多个Gal4的结合位点（UAS）的控制下的质粒电穿孔在受精后24-28小时（HPF）的前脑的前结束。虽然这种方法采用质粒DNA进入前脑的多个地区，GFP的表达诱导transgenically表达的KalTA4转录因子的细胞。因此，通过使用的GA080_9转基因株系，这种做法导致GFP的表达只在发展中国家的嗅球。 GFP的表达，通过这种方法有针对性的细胞呈典型的神经轴突的预测，10嗅球僧帽细胞。这种方法也可用于有针对性地提供其他试剂，包括短发夹RNA干扰表达质粒，这将提供空间和时间上离散的损失函数分析的方法。

Watch this Scientific Journal Video about Targeting Olfactory Bulb Neurons Using Combined In Vivo Electroporation and Gal4-Based Enhancer Trap Zebrafish Lines at JoVE.com

Targeting Olfactory Bulb Neurons Using Combined In Vivo Electroporation and Gal4-Based Enhancer Trap Zebrafish Lines

基因操作的时间和空间分辨率确定频谱的生物现象，他们可以扰乱。在这里，我们使用的时间和空间的离散<em&gt;在体内</em&gt;电结合，与斑马鱼的转基因株系，诱使一个绿色荧光蛋白基因的表达在发展中的嗅球神经元。

针对嗅球神经元采用组合在体内电穿孔和GAL4基于增强陷阱斑马鱼线

In vivo electroporation is a powerful method for delivering DNA expression plasmids, RNAi reagents, and morpholino anti-sense oligonucleotides to specific ...

targeting-olfactory-bulb-neurons-using-combined-vivo-electroporation

Research

JoVE Journal

Neuroscience

Targeting Olfactory Bulb Neurons Using Combined In Vivo Electroporation and Gal4-Based Enhancer Trap Zebrafish Lines

12.0K 次观看.  Pace University. 基因操作的时间和空间分辨率确定频谱的生物现象，他们可以扰乱。在这里，我们使用的时间和空间的离散在体内电结合，与斑马鱼的转基因株系，诱使一个绿色荧光蛋白基因的表达在发展中的嗅球神经元。

视频: Targeting Olfactory Bulb Neurons Using Combined In Vivo Electroporation and Gal4-Based Enhancer Trap Zebrafish Lines

Live Imaging of Innate Immune and Preneoplastic Cell Interactions Using an Inducible Gal4/UAS Expression System in Larval Zebrafish Skin

Here we describe a method to conditionally induce epithelial cell transformation by the use of the 4-Hydroxytamoxifen (4-OHT) inducible KalTA4-ERT2/UAS expression system1 in zebrafish larvae, and the subsequent live imaging of innate immune cell interaction with HRASG12V expressing skin cells. The KalTA4-ERT2/UAS system is both inducible and reversible which allows us to induce cell transformation with precise temporal/spatial resolution in vivo. This provides us with a unique opportunity to live image how individual preneoplastic cells interact with host tissues as soon as they emerge, then follow their progression as well as regression. Recent studies in zebrafish larvae have shown a trophic function of innate immunity in the earliest stages of tumorigenesis2,3. Our inducible system would allow us to live image the onset of cellular transformation and the subsequent host response, which may lead to important insights on the underlying mechanisms for the regulation of oncogenic trophic inflammatory responses. We also discuss how one might adapt our protocol to achieve temporal and spatial control of ectopic gene expression in any tissue of interest.

Studying the earliest events of preneoplastic cell progression and innate immune cell interaction is pivotal to understand and treat cancer. Here we describe a method to conditionally induce epithelial cell transformations and the subsequent live imaging of innate immune cell interaction with HRASG12V expressing skin cells in zebrafish larvae.

先天免疫和癌前细胞间的相互作用使用可诱导的Gal4 / UAS表达系统在斑马鱼幼体皮肤实时成像

Here we describe a method to conditionally induce epithelial cell transformation by the use of the 4-Hydroxytamoxifen (4-OHT) inducible KalTA4-ERT2/UAS ...

科研

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

Optogenetic Activation of Zebrafish Somatosensory Neurons using ChEF-tdTomato

Larval zebrafish are emerging as a model for describing the development and function of simple neural circuits. Due to their external fertilization, rapid development, and translucency, zebrafish are particularly well suited for optogenetic approaches to investigate neural circuit function. In this approach, light-sensitive ion channels are expressed in specific neurons, enabling the experimenter to activate or inhibit them at will and thus assess their contribution to specific behaviors. Applying these methods in larval zebrafish is conceptually simple but requires the optimization of technical details. Here we demonstrate a procedure for expressing a channelrhodopsin variant in larval zebrafish somatosensory neurons, photo-activating single cells, and recording the resulting behaviors. By introducing a few modifications to previously established methods, this approach could be used to elicit behavioral responses from single neurons activated up to at least 4 days post-fertilization (dpf). Specifically, we created a transgene using a somatosensory neuron enhancer, CREST3, to drive the expression of the tagged channelrhodopsin variant, ChEF-tdTomato. Injecting this transgene into 1-cell stage embryos results in mosaic expression in somatosensory neurons, which can be imaged with confocal microscopy. Illuminating identified cells in these animals with light from a 473 nm DPSS laser, guided through a fiber optic cable, elicits behaviors that can be recorded with a high-speed video camera and analyzed quantitatively. This technique could be adapted to study behaviors elicited by activating any zebrafish neuron. Combining this approach with genetic or pharmacological perturbations will be a powerful way to investigate circuit formation and function.

Optogenetic techniques have made it possible to study the contribution of specific neurons to behavior. We describe a method in larval zebrafish for activating single somatosensory neurons expressing a channelrhodopsin variant (ChEF) with a diode-pumped solid state (DPSS) laser and recording the elicited behaviors with a high-speed video camera.

Optogenetic激活斑马鱼的体感神经元使用厨师tdTomato的

Larval  zebrafish are emerging as a model for describing the development and function  of simple neural circuits. Due to their  external fertilization, ...

Micromanipulation of Gene Expression in the Adult Zebrafish Brain Using Cerebroventricular Microinjection of Morpholino Oligonucleotides

Manipulation of gene expression in tissues is required to perform functional studies. In this paper, we demonstrate the cerebroventricular microinjection (CVMI) technique as a means to modulate gene expression in the adult zebrafish brain. By using CVMI, substances can be administered into the cerebroventricular fluid and be thoroughly distributed along the rostrocaudal axis of the brain. We particularly focus on the use of antisense morpholino oligonucleotides, which are potent tools for knocking down gene expression in vivo. In our method, when applied, morpholino molecules are taken up by the cells lining the ventricular surface. These cells include the radial glial cells, which act as neurogenic progenitors. Therefore, knocking down gene expression in the radial glial cells is of utmost importance to analyze the widespread neurogenesis response in zebrafish, and also would provide insight into how vertebrates could sustain adult neurogenesis response. Such an understanding would also help the efforts for clinical applications in human neurodegenerative disorders and central nervous system regeneration. Thus, we present the cerebroventricular microinjection method as a quick and efficient way to alter gene expression and neurogenesis response in the adult zebrafish forebrain. We also provide troubleshooting tips and other useful information on how to carry out the CVMI procedure.

In this article, we demonstrate a method for manipulation of gene expression in the ventricular cells of the adult zebrafish telencephalon using antisense morpholino oligonucleotides. We present this method as an efficient and quick protocol that can be used for functional studies in the adult vertebrate brain.

显微吗啉代寡核苷酸脑室微量注射成年斑马鱼大脑中的基因表达

Manipulation of gene expression in tissues is  required to perform functional studies. In this paper, we demonstrate the  cerebroventricular ...

Electroporation Method for In Vivo Delivery of Plasmid DNA in the Adult Zebrafish Telencephalon

Electroporation is a transfection method in which an electrical field is applied to cells to create temporary pores in a cell membrane and increase its permeability, thereby allowing different molecules to be introduced to the cell. In this paper, electroporation is used to introduce plasmids to ependymoglial cells, which line the ventricular zone of the adult zebrafish telencephalon. A fraction of these cells shows stem cell properties and generates new neurons in the zebrafish brain; therefore, studying their behavior is essential to determine their roles in neurogenesis and regeneration. The introduction of plasmids via electroporation enables long-term labeling and tracking of a single ependymoglial cell. Furthermore, plasmids such as Cre recombinase or Cas9 can be delivered to single ependymoglial cells, which enables gene recombination or gene editing and provides a unique opportunity to assess the cell&#8217;s autonomous gene function in a controlled, natural environment. Finally, this detailed, step-by-step electroporation protocol is used to obtain successful introduction of plasmids into a large number of single ependymoglial cells.

Presented here is an electroporation method for plasmid DNA delivery and ependymoglial cell labeling in the adult zebrafish telencephalon. This protocol is a quick and efficient method to visualize and trace individual ependymoglial cells and opens up new possibilities to apply electroporation to a broad range of genetic manipulations.

成年斑马鱼端脑中疟原虫DNA在活体传递的电穿孔方法

Electroporation is a transfection method in which an electrical field is applied to cells to create temporary pores in a cell membrane and increase its ...

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 ...

生物学

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 ...

Gene Trapping Using Gal4 in Zebrafish

Large clutch size and external development of optically transparent embryos make zebrafish an exceptional vertebrate model system for in vivo insertional mutagenesis using fluorescent reporters to tag expression of mutated genes. Several laboratories have constructed and tested enhancer- and gene-trap vectors in zebrafish, using fluorescent proteins, Gal4- and lexA- based transcriptional activators as reporters 1-7. These vectors had two potential drawbacks: suboptimal stringency (e.g. lack of ability to differentiate between enhancer- and gene-trap events) and low mutagenicity (e.g. integrations into genes rarely produced null alleles). Gene Breaking Transposon (GBTs) were developed to address these drawbacks 8-10. We have modified one of the first GBT vectors, GBT-R15, for use with Gal4-VP16 as the primary gene trap reporter and added UAS:eGFP as the secondary reporter for direct detection of gene trap events. Application of Gal4-VP16 as the primary gene trap reporter provides two main advantages. First, it increases sensitivity for genes expressed at low expression levels. Second, it enables researchers to use gene trap lines as Gal4 drivers to direct expression of other transgenes in very specific tissues. This is especially pertinent for genes with non-essential or redundant functions, where gene trap integration may not result in overt phenotypes. The disadvantage of using Gal4-VP16 as the primary gene trap reporter is that genes coding for proteins with N-terminal signal sequences are not amenable to trapping, as the resulting Gal4-VP16 fusion proteins are unlikely to be able to enter the nucleus and activate transcription. Importantly, the use of Gal4-VP16 does not pre-select for nuclear proteins: we recovered gene trap mutations in genes encoding proteins which function in the nucleus, the cytoplasm and the plasma membrane.

This protocol describes the method of gene trap insertional mutagenesis using Gal4-VP16 as the primary reporter and GFP/RFP as secondary reporters in zebrafish. Approximately one in ten high-expressing F0 fish yield gene trap progeny co-expressing GFP and RFP. The screening procedure can be readily scaled to adapt to the size of the laboratory performing the insertional mutagenesis screen.

基因捕获斑马鱼中使用的Gal4

Large clutch size and external development of optically transparent embryos make zebrafish an exceptional vertebrate model system for in vivo insertional ...

In Vivo Confocal Fluorescence Imaging of Neural Activity Induced by Sensory Stimulation in Partially Restrained Larval Zebrafish

Zebrafish larvae are a promising vertebrate model system for studying the neural mechanisms of behavior. Their translucence and relatively simple neural circuitry facilitate the use of optogenetic techniques in cellular analyses of behavior. Fluorescent indicators of in vivo neural activity, such as GCaMP6s, have been widely used to study the neural activity associated with simple behaviors in larval zebrafish. Here, we present a protocol for detecting sensory-induced activity in semi-restrained zebrafish larvae using the transgenic line Tg(elav3:GCaMP6s). In particular, we use the chemical agent allyl isothiocyanate to induce a robust, reproducible fluorescent response in a brain region at the border of the hindbrain and spinal cord. We discuss the potential uses of GCaMP6s for optical monitoring of neural activity during a range of behavioral paradigms and the limitations of this technique. Our protocol outlines an accessible approach for monitoring dynamic, behavior-related in vivo neural activity in the larval zebrafish brain.

Here, we present a detailed protocol to examine neural activity in brain regions of transgenic zebrafish that express GCaMP calcium indicators using confocal microscopy.

Zebrafish larvae are a promising vertebrate model system for studying the neural mechanisms of behavior. Their translucence and relatively simple neural ...

Zebrafish Optogenetics: Activating Genetically Modified Somatosensory Neuron to Study Larval Behavioral Responses

Source: Palanca A. M. S., et al. <a href="https://www.jove.com/v/50184/" target="_blank">Optogenetic Activation of Zebrafish Somatosensory Neurons using ChEF-tdTomato</a>. J. Vis. Exp. (2013).
This video describes optogenetic techniques by activating genetically modified somatosensory neurons and recording elicited behavioral response with a high-speed video camera.

斑马鱼光遗传学:激活转基因体感神经元以研究幼虫行为反应

Source: Palanca A. M. S., et al. Optogenetic Activation of Zebrafish Somatosensory Neurons using ChEF-tdTomato. J. Vis. Exp. (2013).
This video describes ...