Name: laser-assisted cytoplasmic microinjection in livestock zygotes
Uploaded: 2016-10-05T14:00:00.000+00:00
Duration: 8 min 59 s
Description: Watch this Scientific Journal Video about Laser-assisted Cytoplasmic Microinjection in Livestock Zygotes at JoVE.com

Work related to this technique is supported by NIH/NICHD RO1 HD070044 and USDA/NIFA Hatch projects W-3171 and W-2112.

1，微管生产<ol><li>注射微量<ol><li>放置一个硼硅玻璃毛细管（外径（OD）:1.0毫米，内径（ID）0.75毫米）的一个微量拉马（在右侧和左侧毛细管持有者的中心）并锁定。 </li><li>使用适当的程序来拉玻璃毛细管，因此导致了薄带尖长攻丝机。 （例如:热量:825;拉:30;速度:120;时间:200;压力:500）。 </li><li>小心地从设备中取出拉移液器，将它放在一个microforge在水平位置。 </li><li>带上拉吸管和其玻璃珠成焦点microforge加热器丝。使用目镜标线片以测量所需的厚度和水平移动吸移管，直到加热器灯丝达到目标内径（5微米）。 </li><li>调整热至约45％，轻轻地使吸管向下使其接触灯丝。简要地激活加热器。该W病略微熔融吸移管，因此粘附于灯丝并在冷却，移液器将在接触点产生的直切断裂。 注意:设置适当的温度是在该步骤中的关键，因为温度太高会使吸管弯曲，因此直切是不可能的，温度太低会不足以熔化移液器（ 图1A）。 </li><li>通过从加热器灯丝定位它约10μm距离使吸管尖端附近的近似30°角。设定温度至60％，并激活加热器。 注意:这将弯曲吸管过灯丝。这个角度是所希望的，以便当安装到喷射器（ 图1B）的针的尖端平行于注入板的表面上。 </li><li>手柄显微注射移液器要小心，因为他们都非常尖锐和脆弱。 </li></ol></li><li>保持微量<ol><li>放置borosilic吃玻璃毛细管（外径:1.0mm时，ID:0.75mm）组合在一个微量拉马（在右侧和左侧毛细管持有者的中心）并锁定。 </li><li>使用适当的程序来拉玻璃毛细管，因此导致了薄尖的长锥，甚至和并行墙（例如:热量:815;拉:20;速度:140;时间:175;压力:200）。 </li><li>小心地从牵引装置取出拉出吸管，并将其放置在microforge夹持在水平位置。调整焦距加热器灯丝和吸管。 </li><li>使用目镜标线片来测量移液管的直径和，直到它的直径比灯丝达到180微米移动吸移管。 </li><li>在指定大小，标示用金刚石尖笔的玻璃毛细管，然后轻轻按上拉尖打碎玻璃。这将导致直切（ 图1C）。 </li><li>移动吸管，其尖端靠近灯丝垂直位置。设置温度<html>的microforge至60％。 </li><li>火抛光使用标准技术7直到达到40微米的ID的吸管尖。使用目镜标线检查ID（ 图1D）。 </li><li>使对吸移管的角度。 <ol><li>使吸管回到水平位置约10微米，从灯丝5毫米从其前端远程。设定温度至约60％，并激活加热器弯腰灯丝吸管。直到达到（约30°）所需的角度继续加热。检查视觉角度（ 图1E）。 注意:移液器通常被安装到微量在60°相对于该注射盘底部的近似角度。吸移管的前端弯曲，以便其平行于盘，这是必要的在其中间平面上的正确的注射的胚胎的底部。 </li></ol></li></ol></li></ol>乐"&gt; 2。安装微操作手<ol><li>检查microinjectors满载油和有在系统中没有气泡（在微量气泡防止喷射的精细控制）。 </li><li>检查显微处于中心位置。这将允许广泛的吸移管的移动。 </li><li>插入保持吸管进入左显微保持器和注射针进入右显微操作支架。 </li><li>允许通过油毛细现象进入移液器和检查系统通过使用微量控制吸管内上下移动油正常工作。 注意:如果在针内没有油的运动，有可能是一个障碍。在这种情况下，使用一个新的针。 </li><li>使用显微控制以使移液管进入视显微镜的场的中心。使用放大4倍，检查微量提示是在正确的角度（Parallel到培养皿的底部）。 注:针的正确的设置是一个成功的注入和结果一致性的关键。 </li><li>校准按照制造商的校准手册的激光系统。 </li></ol> 3.注射液盘的制备（图2） <ol><li>放置温热（37℃）SOF-HEPES补充有20％胎牛血清（FBS）的上100毫米培养皿的盖的中心（在材料部分详述组合物）的50微升的下降。放置1 - 将溶液的2微升滴被注入靠近喷射滴。确保胚胎不冷静下来RT以下。 注意:在这个协议中，我们将在葡聚糖红色染料加入到注入溶液，以便能够直观注射部位和以后跟踪。 </li><li>覆盖使用大约10mL，矿物油中的喷射板的液滴。 </li><li>广场上的倒置显微镜阶段注射的菜，并把点子ettes到注射降。检查针是处于焦点的下降中。如使用显微控制需要调整高度。 </li><li>在喷射液滴的上侧 - （20小时后的受精（HPF）17） - 用一个微型分配器，装入约20 30受精卵。因此在注射滴胚的数目由在该人可以注入其中的速度决定于室温下进行注射。别比能够在30分钟内被注入的那些加载更多的胚胎。 <ol><li>要加载的胚胎成微型分配器，完全踩下柱塞和沉浸的尖端插入含有胚胎的解决方案。触摸胚胎被拾起与玻璃毛细管的尖端（一个在时间）和缓慢释放所述柱塞，以便每个胚进入毛细管。重复该步骤直至所有的胚胎都拿起或微型分配器容量已满。 </li><li>以释放加载的胚胎，轻轻压下柱塞，直到整个体积共ntaining胚胎从毛细管释放。 </li></ol></li></ol> 4.显微注射<ol><li>使用4X物镜，加载溶液通过施加负压（吸气）被注入到注射针。装载足够的解决方案注入2 - 3受精卵。然后移动到注射降。 </li><li>在注射下降，移动控股吸管使尖端获得接近合子。与保持微量施加负压（抽吸），以使受精卵被固定到保持吸管和变化到20X物镜。 </li><li>一旦合子附着到保持吸管，用注射吸管轻轻来回移动胚胎不拆卸它检查其质量。良好的品质受精卵应该有两个极体（尽管有时只有一个是看到），并均匀细胞质中。不要注入受精卵异常（ 图3A）。 </li><li>如果有必要，重新定位胚胎一个适当的注射位置。一个良好的定位将是在其中存在的ZP和PM之间的一些空间，所以在PM没有得到由激光损坏。 </li><li>检查注射针是通过轻触注射部位的受精卵定位于胚胎的中面。如果不是在它的中平面，针将趋向于使胚胎旋转代替穿刺。根据需要进行调整的注射针的高度。 </li><li>使用激光（ 图3B）中的ZP的孔。 <ol><li>使用激光的软件发生在那里的孔将作出（在相反侧，其中胚胎附着到保持吸管）的ZP激光掩模版。使用火按钮激光控制窗口中点击。确保该孔的尺寸足够大，以创建在ZP为所述针的开口以不损坏对PM穿过（实施例:0.662毫秒脉冲宽度/ 6.9微米孔大小）。 </li></ol></li><li>通过注射针通过在ZP的孔，使与PM的接触。继续推动吸管向前直到针是¾的方式进入胚胎（ 图3C）。 </li><li>观察在溶液油相间的弯液面的位置，因为这将被用于一致地控制注射体积。 </li><li>适用于注射针，这将导致在PM和细胞质被吸入注射针负压（抽吸）。继续直到细胞质的移动进针加速或当细胞质内容与注射溶液混合起来可看出。这些将指示在PM（ 图3D）的破损。 </li><li>注入回胞质内容随后将溶液放入合子通过施加正压力（注入）细胞质中，直到在溶液油相间的弯液面已经前进一个受精卵的直径相当于过去的起点。 注意:在我们的条件下，这种repre货物内大约7注射液（ 图3E）的复数。 </li><li>变化到4X物镜并移动所注入的胚胎/ s到喷射滴的下侧。通过对控股微量施加正压力释放胚胎。保持在上侧的未注射的胚胎和下降的下侧注入的那些，以帮助跟踪注入/未注射的胚胎和有效地工作。 </li></ol> 5.胚胎恢复和注射结果<ol><li>建立一个新的菜预热SOF-HEPES约200微升3滴。收集用微型分配器（如上所述）的注射的胚胎。通过3 SOF-HEPES移动它们洗净滴注入胚胎。 </li><li>伯爵在注射溶解胚胎并丢弃它们。 注:因为第一个出现半透明，填写整个ZP裂解的受精卵可以从一个完整一是容易区分，它通常具有细胞质内容泄漏到胚胎的外部。 </li><li>或者，用荧光显微镜检查显微注射效率。要知道，紫外线照射可诱发胚胎损害，可能会影响后续发展。 </li><li>在补充有4毫克/ ml牛血清白蛋白（BSA）根据矿物油钾单纯优化介质的50微升液滴（KSOM）25注射的胚胎的培养基在38.5℃，5％CO 2的空气中，并且湿度饱和。 </li><li>补充有5％FBS培养基中注射后两天。 </li><li>检查囊胚（BL）率注射后7天。计算BL速率胚泡胚胎/在于滴培养的胚胎的总数的总数量。 </li></ol>

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The authors have nothing to disclose.

受精卵显微注射是引入解决方案集成到哺乳动物胚胎一套行之有效的方法。与一些依赖于物种和实验的目的变化，这种技术可以广泛地使用。我们展示了如何使用激光来帮助一个钝头微量的入口处进行显微注射胞浆内。某些牲畜物种（如牛，羊，和猪）的合子有黑暗的细胞质中，阻碍注射针的可视化一次胚胎内。此外，他们的血浆膜是非常有弹性的，使得与一个斜面尖刺针（通常用于注入啮齿动?...

1 - 细胞胚胎显微注射细胞质是一个非常强大的技术。它可用于递送任何溶液进入胚胎，例如，产生基因敲除研究基因功能或产生基因编辑动物。农业最相关的农场动物受精卵具有非常高的脂肪酸组合物，使得它们的细胞质不透明和暗1。他们也有相当的弹性质膜（PM）。这些特点使显微注射用常规的原核/胞浆注射在啮齿类动物具有挑战性，往往不准确使用。 细胞质微注射有超过原核显微注射法的优点，因为它是更容易执行并且也导致了注射的胚胎损伤小，导致更高的2生存能力。此协议的总体目标是演示提供解决方案到农场动物受精卵的细胞质一种成功的方法。到能够执行对家畜胚胎效率高细胞质微注射，激光被用来产生在透明带（ZP），然后一个钝端部玻璃针用于显微注射的孔。这一战略旨在减少注射时印在胚胎的机械损伤。然后，注射针内胞质内容抽吸允许PM的效率和自信破损确保溶液被输送到胚胎的细胞质中。 这种技术已在牛胚胎被成功地用于siRNA递送到合子的细胞质3,4和生成使用群集定期相互间隔短回文重复序列（CRISPR）/ CRISPR相关联的系统9（Cas9）系统5的突变。它也适合（稍加修改）注入牛卵丘封闭的卵母细胞6。这里，我们描述我们的注射方案提供的染料，这可以是适用于注射任何宫IRED溶液注入受精卵，并表明，使用这种技术使得最小裂解并且不影响早期胚胎发育。

<table><tbody><tr><td>Micropipette puller</td><td>Sutter Instrument</td><td>P-97</td><td></td></tr><tr><td>Glass capillary</td><td>Sutter instruments</td><td>B100-75-10</td><td>These capillaries are used for making the holding and injecting pipettes. Any thick/standard wall borosilicate tubing without filament can be used.</td></tr><tr><td>Microforge</td><td>Narishige</td><td>MF-9</td><td>Equipped with 10X magnification lense.</td></tr><tr><td>Micromanipulator</td><td>Nikon/ Narishige</td><td>NT88-V3</td><td></td></tr><tr><td>Inverted microscope</td><td>Nikon</td><td>TE2000-U</td><td>Equipped with 4X, 20X lenses and with a laser system.</td></tr><tr><td>Laser</td><td>Research Instruments</td><td>7-47-500</td><td>Saturn 5 Active laser.</td></tr><tr><td>Microdispenser</td><td>Drummond</td><td>3-000-105</td><td>The microdispenser is used to move the embryos. A p10 pipette can also be used but loading as minimal volume as possible.</td></tr><tr><td>100 x 15 mm culture dish</td><td>Falcon</td><td>351029</td><td>Use the lid of the dish to make the injection plate since they have lower walls and will make positioning and moving of the micromanipulator easier. The lid of a 60 mm culture dish can also be used.</td></tr><tr><td>35 mm culture dish</td><td>Corning</td><td>430165</td><td>These dishes are used for culturing the embryos in 50 &amp;mu;l drops covered with mineral oil. Alternatively, a 4 well dish can also be used. Regardless of the dish chosen to culture the embryos, they always have to be equilibrated in the incubator for at least 4 hr prior to transfering the embryos to them.</td></tr><tr><td>Incubator</td><td>Sanyo</td><td>MCO-19AIC</td><td>Any incubator that can be set to 38.5 &amp;deg;C, 5% CO&lt;sub&gt;2&lt;/sub&gt; conditions can be used.</td></tr><tr><td>Stereomicroscope</td><td>Nikon</td><td>SMZ800</td><td>Used for visualizing the embryos in the culture drops and during washes. Any stereomicroscope with a 10X magnification can be used.</td></tr><tr><td>Control Unit HT</td><td>Minitube</td><td>12055/0400</td><td>Heating system attached to the stereomicroscope.</td></tr><tr><td>Heated Microscope Stage</td><td>Minitube</td><td>12055/0003</td><td>Heating system attached to the stereomicroscope.</td></tr><tr><td>Dextran-Red</td><td>Thermo Scientific</td><td>D1828</td><td>A sterile 10 mg/ml solution is used to inject.</td></tr><tr><td>Mineral Oil</td><td>sigma</td><td>M8410</td><td>Keep the mineral oil at room temperature and&amp;nbsp; protected from light using foil paper.</td></tr><tr><td>KSOMaa Evolve Bovine</td><td>Zenit</td><td>ZEBV-100</td><td>Supplemented with 4 mg/ml BSA. KSOM plates for embryo culture should be equilibrated in an incubator for at least 4 hr before use.</td></tr><tr><td>FBS</td><td>Gemini-Bio</td><td>100-525</td><td>Use a stem-cell qualified FBS.</td></tr><tr><td>Zygotes</td><td></td><td></td><td>Zygotes are injected 17 - 20 hpf and can be &lt;em&gt;in-vitro&lt;/em&gt;- or &lt;em&gt;in-vivo&lt;/em&gt;-derived.</td></tr><tr><td>NaCl</td><td>Sigma</td><td>S5886</td><td>Final concentration: 107.7 mM. Component of SOF-HEPES medium.</td></tr><tr><td>KCl</td><td>Sigma</td><td>P5405</td><td>Final concentration: 7.16 mM. Component of SOF-HEPES medium.</td></tr><tr><td>KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;</td><td>Sigma</td><td>P5655</td><td>Final concentration: 1.19 mM. Component of SOF-HEPES medium.</td></tr><tr><td>MgCl&lt;sub&gt;2&lt;/sub&gt; 6H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Sigma</td><td>M2393</td><td>Final concentration: 0.49 mM. Component of SOF-HEPES medium.</td></tr><tr><td>Sodium DL-lactate</td><td>Sigma</td><td>L4263</td><td>Final concentration: 5.3 mM. Component of SOF-HEPES medium.</td></tr><tr><td>CaCl&lt;sub&gt;2&lt;/sub&gt;-2H&lt;sub&gt;2&lt;/sub&gt;O&amp;nbsp;</td><td>Sigma</td><td>C7902</td><td>Final concentration: 1.71 mM. Component of SOF-HEPES medium.</td></tr><tr><td>D-(&amp;minus;)-Fructose&amp;nbsp;</td><td>Sigma</td><td>F3510</td><td>Final concentration: 0.5 mM. Component of SOF-HEPES medium.</td></tr><tr><td>HEPES&amp;nbsp;</td><td>Sigma</td><td>H4034</td><td>Final concentration: 21 mM. Component of SOF-HEPES medium.</td></tr><tr><td>MEM-NEAA</td><td>Sigma</td><td>M7145</td><td>Final concentration: 1x. Component of SOF-HEPES medium.</td></tr><tr><td>BME-EAA</td><td>Sigma</td><td>B6766</td><td>Final concentration: 1x. Component of SOF-HEPES medium.</td></tr><tr><td>NaHCO&lt;sub&gt;3&lt;/sub&gt;</td><td>Sigma</td><td>S5761</td><td>Final concentration: 4 mM. Component of SOF-HEPES medium.</td></tr><tr><td>Sodium pyruvate</td><td>Sigma</td><td>P4562</td><td>Final concentration: 0.33 mM. Component of SOF-HEPES medium.</td></tr><tr><td>Glutamax</td><td>Gibco</td><td>35050</td><td>Final concentration: 1 mM. Component of SOF-HEPES medium.</td></tr><tr><td>BSA</td><td>Sigma</td><td>A-3311</td><td>Final concentration: 1 mg/ml. Component of SOF-HEPES medium.</td></tr><tr><td>Gentamicin</td><td>Sigma</td><td>G-1397</td><td>Final concentration: 5 &amp;mu;g/ml. Component of SOF-HEPES medium.</td></tr><tr><td>Water for embryo transfer</td><td>Sigma</td><td>W1503</td><td>Component of SOF-HEPES medium.</td></tr><tr><td>SOF-HEPES medium</td><td>Made in the lab</td><td></td><td>pH 7.3 - 7.4, 280 &amp;plusmn; 10 mOs. Filter sterilized through a 22 &amp;mu;m filter can be stored in the fridge at 4 &amp;deg;C for 1 month. Warm in 37 &amp;deg;C water bath before use.</td></tr></tbody></table>

laser-assisted cytoplasmic microinjection in livestock zygotes

Cytoplasmic microinjection into one-cell embryos is a very powerful technique. As an example, it enables the delivery of genome editing tools that can create genetic modifications that will be present in every cell of an adult organism. It can also be used to deliver siRNA, mRNAs or blocking antibodies to study gene function in preimplantation embryos. The conventional technique for microinjecting embryos used in rodents consists of a very thin micropipette that directly penetrates the plasma membrane when advanced into the embryo. When this technique is applied to livestock animals it usually results in low efficiency. This is mainly because in contrast to mice and rats, bovine, ovine, and porcine zygotes have a very dark cytoplasm and a highly elastic plasma membrane that makes visualization during injection and penetration of the plasma membrane hard to achieve. In this protocol, we describe a suitable microinjection method for the delivery of solutions into the cytoplasm of cattle zygotes that has proved to be successful for sheep and pig embryos as well. First, a laser is used to create a hole in the zona pellucida. Then a blunt-end glass micropipette is introduced through the hole and advanced until the tip of the needle reaches about 3/4 into the embryo. Then, the plasma membrane is broken by aspiration of cytoplasmic content inside the needle. Finally, the aspirated cytoplasmic content followed by the solution of interest is injected back into the embryonic cytoplasm. This protocol has been successfully used for the delivery of different solutions into bovine and ovine zygotes with 100% efficiency, minimal lysis, and normal blastocysts development rates.

激光辅助显微注射细胞质是一个强大而可靠的协议，以提供解决方案为牲畜受精卵的细胞质中。 图3显示了受精卵的大致轮廓之前和注射，以及该技术的整体轮廓之后。葡聚糖红被用作注射溶液，以允许注入和注入效率和准确度的跟踪点。溶液的成功递送在图4中示出表示最近注入胚胎，其中染料被均匀地分布在细胞质中。使用这种技术的胚胎的100...

Watch this Scientific Journal Video about Laser-assisted Cytoplasmic Microinjection in Livestock Zygotes at JoVE.com

Laser-assisted Cytoplasmic Microinjection in Livestock Zygotes

This protocol shows how to perform cytoplasmic microinjection in farm animal zygotes. This technique can be used to deliver any solution into the one-cell embryo such as genome editing tools to generate knockout animals.

激光辅助畜禽受精卵细胞质显微注射

Cytoplasmic microinjection into one-cell embryos is a very powerful technique. As an example, it enables the delivery of genome editing tools that can ...

laser-assisted-cytoplasmic-microinjection-in-livestock-zygotes

Research

JoVE Journal

Genetics

8.9K 次观看.  University of California, Davis. This protocol shows how to perform cytoplasmic microinjection in farm animal zygotes. This technique can be used to deliver any solution into the one-cell embryo such as genome editing tools to generate knockout animals.

视频: Laser-assisted Cytoplasmic Microinjection in Livestock Zygotes

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

Delivery of Nucleic Acids through Embryo Microinjection in the Worldwide Agricultural Pest Insect, Ceratitis capitata

The Mediterranean fruit fly (medfly) Ceratitis capitata (Wiedemann) (Diptera: Tephritidae) is a pest species with extremely high agricultural relevance. This is due to its reproductive behavior: females damage the external surface of fruits and vegetables when they lay eggs and the hatched larvae feed on their pulp. Wild C. capitata populations are traditionally controlled through insecticide spraying and/or eco-friendly approaches, the most successful being the Sterile Insect Technique (SIT). The SIT relies on mass-rearing, radiation-based sterilization and field release of males that retain their capacity to mate but are not able to generate fertile progeny. The advent and the subsequent rapid development of biotechnological tools, together with the availability of the medfly genome sequence, has greatly boosted our understanding of the biology of this species. This favored the proliferation of new strategies for genome manipulation, which can be applied to population control.
In this context, embryo microinjection plays a dual role in expanding the toolbox for medfly control. The ability to interfere with the function of genes that regulate key biological processes, indeed, expands our understanding of the molecular machinery underlying medfly invasiveness. Furthermore, the ability to achieve germ-line transformation facilitates the production of multiple transgenic strains that can be tested for future field applications in novel SIT settings. Indeed, genetic manipulation can be used to confer desirable traits that can, for example, be used to monitor sterile male performance in the field, or that can result in early life-stage lethality. Here we describe a method to microinject nucleic acids into medfly embryos to achieve these two main goals.

Delivery of Nucleic Acids through Embryo Microinjection in the Worldwide Agricultural Pest Insect, Ceratitis capitata

The Mediterranean fruit fly (medfly) Ceratitis capitata (Diptera: Tephritidae) is a worldwide pest of agriculture. A deeper understanding of its biology is key to control medfly populations and thus reduce economic impact. Embryo microinjection is a fundamental tool allowing both germ-line transformation and reverse genetics studies in this species.

核酸的传递通过显微注射胚胎在世界范围内的农业害虫，<em&gt;地中海实蝇</em

The Mediterranean fruit fly (medfly) Ceratitis capitata (Wiedemann) (Diptera: Tephritidae) is a pest species with extremely high agricultural relevance. ...

遗传学

Microinjection of mRNA and Morpholino Antisense Oligonucleotides in Zebrafish Embryos.

An essential tool for investigating the role of a gene during development is the ability to perform gene knockdown, overexpression, and misexpression studies. In zebrafish (Danio rerio), microinjection of RNA, DNA, proteins, antisense oligonucleotides and other small molecules into the developing embryo provides researchers a quick and robust assay for exploring gene function in vivo. In this video-article, we will demonstrate how to prepare and microinject in vitro synthesized EGFP mRNA and a translational-blocking morpholino oligo against pkd2, a gene associated with autosomal dominant polycystic kidney disease (ADPKD), into 1-cell stage zebrafish embryos. We will then analyze the success of the mRNA and morpholino microinjections by verifying GFP expression and phenotype analysis. Broad applications of this technique include generating transgenic animals and germ-line chimeras, cell-fate mapping and gene screening. Herein we describe a protocol for overexpression of EGFP and knockdown of pkd2 by mRNA and morpholino oligonucleotide injection.

Microinjection is a well-established and effective method for introducing foreign substances into fertilized zebrafish embryos. Here, we demonstrate a robust microinjection technique for performing mRNA overexpression, and morpholino oligonucleotide gene knockdown studies in zebrafish.

显微注射mRNA和斑马鱼的胚胎吗啉反义寡核苷酸。

An essential tool for investigating the role of a gene during development is the ability to perform gene knockdown, overexpression, and misexpression ...

生物学

Microinjection of Zebrafish Embryos to Analyze Gene Function

One of the advantages of studying zebrafish is the ease and speed of manipulating protein levels in the embryo.  Morpholinos, which are synthetic oligonucleotides with antisense complementarity to target RNAs, can be added to the embryo to reduce the expression of a particular gene product.  Conversely, processed mRNA can be added to the embryo to increase levels of a gene product.  The vehicle for adding either mRNA or morpholino to an embryo is microinjection.  Microinjection is efficient and rapid, allowing for the injection of hundreds of embryos per hour.  This video shows all the steps involved in microinjection.  Briefly, eggs are collected immediately after being laid and lined up against a microscope slide in a Petri dish.  Next, a fine-tipped needle loaded with injection material is connected to a microinjector and an air source, and the microinjector controls are adjusted to produce a desirable injection volume.  Finally, the needle is plunged into the embryo's yolk and the morpholino or mRNA is expelled.

This video shows how morpholino or mRNA can be injected into zebrafish embryos at the one-cell stage to decrease or increase the level of specific gene products during subsequent development.

斑马鱼的胚胎显微注射来分析基因的功能

One of the advantages of studying zebrafish is the ease and speed of manipulating protein levels in the embryo.  Morpholinos, which are synthetic ...

Microstructured Devices for Optimized Microinjection and Imaging of Zebrafish Larvae

Zebrafish have emerged as a powerful model of various human diseases and a useful tool for an increasing range of experimental studies, spanning fundamental developmental biology through to large-scale genetic and chemical screens. However, many experiments, especially those related to infection and xenograft models, rely on microinjection and imaging of embryos and larvae, which are laborious techniques that require skill and expertise. To improve the precision and throughput of current microinjection techniques, we developed a series of microstructured devices to orient and stabilize zebrafish embryos at 2 days post fertilization (dpf) in ventral, dorsal, or lateral orientation prior to the procedure. To aid in the imaging of embryos, we also designed a simple device with channels that orient 4 zebrafish laterally in parallel against a glass cover slip. Together, the tools that we present here demonstrate the effectiveness of photolithographic approaches to generate useful devices for the optimization of zebrafish techniques.

Microinjection of zebrafish embryos and larvae is a crucial but challenging technique used in many zebrafish models. Here, we present a range of microscale tools to aid in the stabilization and orientation of zebrafish for both microinjection and imaging.

斑马鱼幼虫显微注射和成像的微装置

Zebrafish have emerged as a powerful model of various human diseases and a useful tool for an increasing range of experimental studies, spanning ...

Zygote Microinjection for Creating Gene Cassette Knock-in and Flox Alleles in Mice

CRISPR-Cas technology has enabled the rapid and effortless generation of genetically modified mice. Specifically, mice and point mutant mice are readily produced by electroporation of CRISPR factors (and single-stranded oligo DNA donors) into the zygote. In contrast, gene cassette (&#62;1 kb) knock-in and floxed mice are mainly generated by microinjection of CRISPR factors and double-stranded DNA donors into zygotes. Genome editing technologies have also increased the flexibility of genetically modified mice production. It is now possible to introduce the intended mutations in the target genomic regions in a number of beneficial inbred mouse strains. Our team has produced over 200 gene cassette knock-in mouse lines, and over 110 floxed mouse lines by zygote microinjection of CRISPR-Cas9 following requests from several countries, including Japan. Some of these genome editing used BALB/c, C3H/HeJ, and C57BL/6N inbred strains, however most used C57BL/6J. Unlike the electroporation method, genome editing by zygote microinjection in various inbred strains of mice is not that easy. However, gene cassette knock-in and floxed mice on single inbred genetic backgrounds are as critical as genetic humanized, fluorescent reporter, and conditional knockout mouse models. Therefore, this article presents the protocol for the zygote microinjection of CRISPR factors and double-stranded DNA donors in C57BL/6J mice for generating gene cassette knock-in and floxed mice. This article exclusively focuses on nuclear injection rather than cytoplasmic injection. In addition to zygote microinjection, we outline the timeline for the production process and peripheral techniques such as induction of superovulation and embryo transfer.

The present protocol describes zygote microinjection of CRISPR-Cas9 and donor DNA to efficiently produce gene cassette knock-in and floxed mice.

受精卵显微注射用于在小鼠中产生基因盒敲入和絮状等位基因

CRISPR-Cas technology has enabled the rapid and effortless generation of genetically modified mice. Specifically, mice and point mutant mice are readily ...

High Throughput Microinjections of Sea Urchin Zygotes

Microinjection into cells and embryos is a common technique that is used to study a wide range of biological processes. In this method a small amount of treatment solution is loaded into a microinjection needle that is used to physically inject individual immobilized cells or embryos. Despite the need for initial training to perform this procedure for high-throughput delivery, microinjection offers maximum efficiency and reproducible delivery of a wide variety of treatment solutions (including complex mixtures of samples) into cells, eggs or embryos. Applications to microinjections include delivery of DNA constructs, mRNAs, recombinant proteins, gain of function, and loss of function reagents. Fluorescent or colorimetric dye is added to the injected solution to enable instant visualization of efficient delivery as well as a tool for reliable normalization of the amount of the delivered solution. The described method enables microinjection of 100-400 sea urchin zygotes within 10-15 min.

Microinjection is a common technique used to deliver DNA constructs, mRNAs, morpholino antisense oligonucleotides or other treatments into eggs, embryos, and cells of various species.

海胆受精卵的高通量显微注射

Microinjection into cells and embryos is a common technique that is used to study a wide range of biological processes. In this method a small amount of ...

Laser-assisted Lentiviral Gene Delivery to Mouse Fertilized Eggs

Lentiviruses are efficient vectors for gene delivery to mammalian cells. Following transduction, the lentiviral genome is stably incorporated into the host chromosome and is passed on to progeny. Thus, they are ideal vectors for creation of stable cell lines, in vivo delivery of indicators, and transduction of single cell fertilized eggs to create transgenic animals. However, mouse fertilized eggs and early stage embryos are protected by the zona pellucida, a glycoprotein matrix that forms a barrier against lentiviral gene delivery. Lentiviruses are too large to penetrate the zona and are typically delivered by microinjection of viral particles into the perivitelline cavity, the space between the zona and the embryonic cells. The requirement for highly skilled technologists and specialized equipment has minimized the use of lentiviruses for gene delivery to mouse embryos. This article describes a protocol for permeabilizing the mouse fertilized eggs by perforating the zona with a laser. Laser-perforation does not result in any damage to embryos and allows lentiviruses to gain access to embryonic cells for gene delivery. Transduced embryos can develop into blastocyst in vitro, and if implanted in pseudopregnant mice, develop into transgenic pups. The laser used in this protocol is effective and easy to use. Genes delivered by lentiviruses stably incorporate into mouse embryonic cells and are germline transmittable. This is an alternative method for creation of transgenic mice that requires no micromanipulation and microinjection of fertilized eggs.

Mouse fertilized eggs and early stage embryos are protected by the zona pellucida, a glycoprotein matrix that forms a barrier against gene delivery. This article describes a protocol for perforating the zona with a laser to transduce embryonic cells with lentiviral vectors and to create transgenic mice.

激光辅助慢病毒基因传递到小鼠受精卵

Lentiviruses are efficient vectors for gene delivery to mammalian cells. Following transduction, the lentiviral genome is stably incorporated into the ...

Zebrafish Microinjection Techniques

One of the major advantages to working with zebrafish (Danio rerio) is that their genetics can be easily manipulated by microinjection of early stage embryos. Using this technique, solutions containing genetic material or knockdown constructs are delivered into the blastomeres: the embryonic cells sitting atop the yolk of the newly fertilized egg. Delivery into the cytoplasm is achieved either through direct injection into the blastomere, or via natural cytoplasmic movements that occur after a solution is injected into the yolk. Successful genetic manipulations are usually followed by quantification of embryonic phenotypes in order to elucidate the genetic mechanisms of development.
This video will provide an introduction to carrying out microinjections in zebrafish embryos. The discussion begins with a review of the essential tools for the technique, including the injection apparatus and the microinjector, which controls fluid movement with pressure pulses of air. Next, important preparatory steps are demonstrated, such as the pouring of agar plates to stabilize embryos during injection and calibration of the microinjection apparatus. The injection procedure is then presented along with tips on when and where injections should be performed. Finally, applications of the microinjection technique are discussed, including gene overexpression via mRNA injection, gene silencing by delivery of antisense morpholino oligonucleotides, and the generation of transgenic zebrafish using specially engineered plasmid DNA.

Introduction to Microinjection of Early Zebrafish Embryos

斑马鱼显微注射技术

One of the major advantages to working with zebrafish (Danio rerio) is that their genetics can be easily manipulated by microinjection of early stage ...

教育

JoVE Science Education

基础生物学

模式生物II:小鼠，斑马鱼和鸡

Laser-Assisted Lentiviral Gene Delivery: A Technique to Permeabilize Mouse Fertilized Eggs to Facilitate Lentiviral Gene Delivery

Source: Martin, N. P., et al. <a href="https://www.jove.com/v/58327">Laser-assisted Lentiviral Gene Delivery to Mouse Fertilized Eggs.</a> J. Vis. Exp. (2018).
In this video, we demonstrate the laser-assisted permeation of the protective layer of zona pellucida in mouse fertilized eggs for facilitating lentiviral gene delivery. Lentivirus enables the generation of transgenic animals with a gene of interest stably integrated into their genome.

激光辅助慢病毒基因递送:一种透化小鼠受精卵以促进慢病毒基因递送的技术

Source: Martin, N. P., et al. Laser-assisted Lentiviral Gene Delivery to Mouse Fertilized Eggs. J. Vis. Exp. (2018).
In this video, we demonstrate the ...