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>

<ol>
	<li>McEvoy, T., Coull, G., Broadbent, P., Hutchinson, J., Speake, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fatty+acid+composition+of+lipids+in+immature+cattle,+pig+and+sheep+oocytes+with+intact+zona+pellucida.">Fatty acid composition of lipids in immature cattle, pig and sheep oocytes with intact zona pellucida.</a> J Reprod Fertil. 118 (1), 163-170 (2000).</li><li>Brinster, R. L., Chen, H. Y., Trumbauer, M. E., Yagle, M. K., Palmiter, R. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Factors+affecting+the+efficiency+of+introducing+foreign+DNA+into+mice+by+microinjecting+eggs.">Factors affecting the efficiency of introducing foreign DNA into mice by microinjecting eggs.</a> Proc Natl Acad Sci U S A. 82 (13), 4438-4442 (1985).</li><li>Ross, P. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Parthenogenetic+activation+of+bovine+oocytes+using+bovine+and+murine+phospholipase+C+zeta.">Parthenogenetic activation of bovine oocytes using bovine and murine phospholipase C zeta.</a> BMC Dev Biol. 8 (1), (2008).</li><li>Canovas, J., Cibelli, J. B., Ross, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Jumonji+domain-containing+protein+3+regulates+histone+3+lysine+27+methylation+during+bovine+preimplantation+development.">Jumonji domain-containing protein 3 regulates histone 3 lysine 27 methylation during bovine preimplantation development.</a> Proc Natl Acad Sci U S A. 109 (7), 2400-2405 (2012).</li><li>Bogliotti, Y. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=4+Developmental+outcomes+and+effiency+of+two+CRISPR/Cas9+microinjection+methods+in+bovine+zygotes.">4 Developmental outcomes and effiency of two CRISPR/Cas9 microinjection methods in bovine zygotes.</a> Reprod Fertil Dev. 27 (1), 94-94 (2015).</li><li>Bakhtari, A., Ross, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DPPA3+prevents+cytosine+hydroxymethylation+of+the+maternal+pronucleus+and+is+required+for+normal+development+in+bovine+embryos.">DPPA3 prevents cytosine hydroxymethylation of the maternal pronucleus and is required for normal development in bovine embryos.</a> Epigenetics. 9 (9), 1271-1279 (2014).</li><li>Yaul, M., Bhatti, R., Lawrence, 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+process+of+polishing+borosilicate+glass+capillaries+used+for+fabrication+of+in-vitro+fertilization+(iVF)+micro-pipettes.">Evaluating the process of polishing borosilicate glass capillaries used for fabrication of in-vitro fertilization (iVF) micro-pipettes.</a> Biomed Microdevices. 10 (1), 123-128 (2008).</li><li>Sutter Instrument. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Pipette Cookbook 2015 P-97 &amp; P-1000 Micropipette Pullers. , (2015).</li><li>Sutter Instrument. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> P-97 Flaming/BrownTM Micropipette Puller Operation Manual Rev. 2.30 - DOM (20140825). , (2014).</li><li>Cibelli, J. B., Lanza, R. P., Campbell, K. H. S., West, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Principles of cloning. , (2002).</li><li>Wang, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+of+a+reporter+gene+after+microinjection+of+mammalian+artificial+chromosomes+into+pronuclei+of+bovine+zygotes.">Expression of a reporter gene after microinjection of mammalian artificial chromosomes into pronuclei of bovine zygotes.</a> Mol Reprod Dev. 60 (4), 433-438 (2001).</li></ol>

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 border="0" cellpadding="0" cellspacing="0" width="786">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:268px;">Micropipette puller</td>
			<td style="width:143px;">Sutter Instrument</td>
			<td style="width:119px;">P-97</td>
			<td style="width:257px;"></td>
		</tr>
		<tr height="80">
			<td height="80" style="height:80px;width:268px;">Glass capillary</td>
			<td style="width:143px;">Sutter instruments</td>
			<td style="width:119px;">B100-75-10</td>
			<td style="width:257px;">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 height="20">
			<td height="20" style="height:20px;width:268px;">Microforge</td>
			<td style="width:143px;">Narishige</td>
			<td style="width:119px;">MF-9</td>
			<td>Equipped with 10X magnification lense.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:268px;">Micromanipulator</td>
			<td style="width:143px;">Nikon/ Narishige</td>
			<td>NT88-V3</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:268px;">Inverted microscope</td>
			<td style="width:143px;">Nikon</td>
			<td>TE2000-U</td>
			<td style="width:257px;">Equipped with 4x, 20x lenses and with a laser system.</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Laser</td>
			<td style="width:143px;">Research Instruments</td>
			<td>7-47-500</td>
			<td>Saturn 5 Active laser.</td>
		</tr>
		<tr height="80">
			<td height="80" style="height:80px;width:268px;">Microdispenser</td>
			<td>Drummond</td>
			<td>3-000-105</td>
			<td style="width:257px;">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 height="100">
			<td height="100" style="height:100px;width:268px;">60mm culture dish</td>
			<td style="width:143px;">Corning</td>
			<td style="width:119px;">430166</td>
			<td style="width:257px;">Use the lid of the dish to make the injection plate since they have lower walls and will make positioning and moving of the micropipettes with the micromanipulator easier.&#160;</td>
		</tr>
		<tr height="160">
			<td height="160" style="height:160px;width:268px;">35mm culture dish</td>
			<td style="width:143px;">Corning</td>
			<td style="width:119px;">430165</td>
			<td style="width:257px;">These dishes are used for culturing the embryos in 50&#956;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 hours prior to transfering the embryos to them.</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:268px;">Incubator</td>
			<td style="width:143px;">Sanyo</td>
			<td style="width:119px;">MCO-19AIC</td>
			<td style="width:257px;">Any incubator that can be set to 38.5&#176;C 5% CO2 conditions can be used.</td>
		</tr>
		<tr height="80">
			<td height="80" style="height:80px;width:268px;">Stereomicroscope</td>
			<td style="width:143px;">Nikon</td>
			<td>SMZ800</td>
			<td style="width:257px;">Used for visualizing the embryos in the culture drops and during washes. Any stereomicroscope with a 10x magnification can be used.</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Control Unit HT</td>
			<td style="width:143px;">Minitube</td>
			<td>12055/0400</td>
			<td style="width:257px;">Heating system attached to the stereomicroscope.</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Heated Microscope Stage</td>
			<td style="width:143px;">Minitube</td>
			<td>12055/0003</td>
			<td style="width:257px;">Heating system attached to the stereomicroscope.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:268px;">Dextran-Red</td>
			<td>Thermo Scientific</td>
			<td>D1828</td>
			<td>A sterile 10mg/ml solution is used to inject.</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:268px;">Mineral Oil</td>
			<td style="width:143px;">sigma</td>
			<td style="width:119px;">M8410</td>
			<td style="width:257px;">Keep the mineral oil at room temperature and&#160; protected from light using foil paper.</td>
		</tr>
		<tr height="80">
			<td height="80" style="height:80px;width:268px;">KSOMaa Evolve Bovine</td>
			<td style="width:143px;">Zenit</td>
			<td style="width:119px;">ZEBV-100</td>
			<td style="width:257px;">Supplemented with 4mg/ml BSA. KSOM plates for embryo culture should be equilibrated in an incubator for at least 4 hours before use.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:268px;">FBS</td>
			<td style="width:143px;">Gemini-Bio</td>
			<td style="width:119px;">100-525</td>
			<td>Use a stem-cell qualified FBS.</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:268px;">Zygotes</td>
			<td style="width:143px;"></td>
			<td style="width:119px;"></td>
			<td style="width:257px;">Zygotes are injected 17-20 hpf and can be in-vitro- or in-vivo-derived.</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:268px;">NaCl</td>
			<td style="width:143px;">Sigma</td>
			<td style="width:119px;">S5886</td>
			<td style="width:257px;">Final concentration: 107.7mM. Component of SOF-HEPES medium.</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:268px;">KCl</td>
			<td style="width:143px;">Sigma</td>
			<td style="width:119px;">P5405</td>
			<td style="width:257px;">Final concentration: 7.16mM. Component of SOF-HEPES medium.</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:268px;">KH2PO4</td>
			<td style="width:143px;">Sigma</td>
			<td style="width:119px;">P5655</td>
			<td style="width:257px;">Final concentration: 1.19mM. Component of SOF-HEPES medium.</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:268px;">MgCL2 6H2O</td>
			<td style="width:143px;">Sigma</td>
			<td style="width:119px;">M2393</td>
			<td style="width:257px;">Final concentration: 0.49mM. Component of SOF-HEPES medium.</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:268px;">Sodium DL-lactate</td>
			<td style="width:143px;">Sigma</td>
			<td style="width:119px;">L4263</td>
			<td style="width:257px;">Final concentration: 5.3mM. Component of SOF-HEPES medium.</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:268px;">CaCl2-2H2O&#160;</td>
			<td style="width:143px;">Sigma</td>
			<td style="width:119px;">C7902</td>
			<td style="width:257px;">Final concentration: 1.71mM. Component of SOF-HEPES medium.</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:268px;">D-(&#8722;)-Fructose&#160;</td>
			<td style="width:143px;">Sigma</td>
			<td style="width:119px;">F3510</td>
			<td style="width:257px;">Final concentration: 0.5mM. Component of SOF-HEPES medium.</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:268px;">HEPES&#160;</td>
			<td style="width:143px;">Sigma</td>
			<td style="width:119px;">H4034</td>
			<td style="width:257px;">Final concentration: 21mM. Component of SOF-HEPES medium.</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:268px;">MEM-NEAA</td>
			<td style="width:143px;">Sigma</td>
			<td style="width:119px;">M7145</td>
			<td style="width:257px;">Final concentration: 1X. Component of SOF-HEPES medium.</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:268px;">BME-EAA</td>
			<td style="width:143px;">Sigma</td>
			<td style="width:119px;">B6766</td>
			<td style="width:257px;">Final concentration: 1X. Component of SOF-HEPES medium.</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:268px;">NaHCO3</td>
			<td style="width:143px;">Sigma</td>
			<td style="width:119px;">S5761</td>
			<td style="width:257px;">Final concentration: 4mM. Component of SOF-HEPES medium.</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:268px;">Sodium pyruvate</td>
			<td style="width:143px;">Sigma</td>
			<td style="width:119px;">P4562</td>
			<td style="width:257px;">Final concentration: 0.33mM. Component of SOF-HEPES medium.</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:268px;">Glutamax</td>
			<td style="width:143px;">Gibco</td>
			<td style="width:119px;">35050</td>
			<td style="width:257px;">Final concentration: 1mM. Component of SOF-HEPES medium.</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:268px;">BSA</td>
			<td style="width:143px;">Sigma</td>
			<td style="width:119px;">A-3311</td>
			<td style="width:257px;">Final concentration: 1mg/ml. Component of SOF-HEPES medium.</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:268px;">Gentamicin</td>
			<td style="width:143px;">Sigma</td>
			<td style="width:119px;">G-1397</td>
			<td style="width:257px;">Final concentration: 5&#956;g/ml. Component of SOF-HEPES medium.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:268px;">Water for embryo transfer</td>
			<td style="width:143px;">Sigma</td>
			<td style="width:119px;">W1503</td>
			<td style="width:257px;">Component of SOF-HEPES medium.</td>
		</tr>
		<tr height="80">
			<td height="80" style="height:80px;width:268px;">SOF-HEPES medium</td>
			<td style="width:143px;">Made in the lab</td>
			<td style="width:119px;"></td>
			<td style="width:257px;">pH 7.3-7.4, 280&#177;10 mOs. Filter sterilized through a 22&#956;m filter can be stored in the fridge at 4&#176; C for 1 month. Warm in 37 &#176;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

siRNA Transfection and EMSA Analyses on Freshly Isolated Human Villous Cytotrophoblasts

Human primary villous cytotrophoblasts are a very useful source of primary cells to study placental functions and regulatory mechanisms, and to comprehend diseases related to pregnancy. In this protocol, human primary villous cytotrophoblasts freshly isolated from placentas through a standard DNase/trypsin protocol are microporated with small interfering RNA (siRNA). This approach provided greater efficiency for siRNA transfection when compared to a lipofection-based method. Transfected cells can subsequently be analyzed by standard Western blot within a time frame of 3-4 days post-transfection. In addition, using cultured primary villous cytotrophoblasts, Electrophoretic Mobility Shift Assay (EMSA) analysis was optimized and performed on extracts from days 1 to 4. The use of these cultured primary cells and the protocol described allow for an evaluation of the implication of specific genes and transcription factors in the process of villous cytotrophoblast differentiation into a syncytiotrophoblast-like cell layer. However, the limited time span allowable in culture precludes the use of methods requiring more time, such as generation of a stable cell population. Therefore testing of this cell population requires highly optimized gene transfer protocols.

This protocol describes a method for efficiently transfecting siRNA in freshly isolated human villous cytotrophoblasts using microporation and identifying DNA-protein complexes in these cells. Transfected cells can be monitored by Western blot and EMSA analyses during the 4-day culture time.

在新鲜分离的人代绒毛滋养细胞转染和EMSA分析

Human primary villous cytotrophoblasts are a very useful source of primary cells to study placental functions and regulatory mechanisms, and to comprehend ...

科研

遗传学

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. ...

Optogenetic Random Mutagenesis Using Histone-miniSOG in C. elegans

Forward genetic screening in model organisms is the workhorse to discover functionally important genes and pathways in many biological processes. In most mutagenesis-based screens, researchers have relied on the use of toxic chemicals, carcinogens, or irradiation, which requires designated equipment, safety setup, and/or disposal of hazardous materials. We have developed a simple approach to induce heritable mutations in C. elegans using germline-expressed histone-miniSOG, a light-inducible potent generator of reactive oxygen species. This mutagenesis method is free of toxic chemicals and requires minimal laboratory safety and waste management. The induced DNA modifications include single-nucleotide changes and small deletions, and complement those caused by classical chemical mutagenesis. This methodology can also be used to induce integration of extrachromosomal transgenes. Here, we provide the details of the LED setup and protocols for standard mutagenesis and transgene integration.

Optogenetic Random Mutagenesis Using Histone-miniSOG in C. elegans

Genetically-encoded histone-miniSOG induces genome-wide heritable mutations in a blue&#160;light-dependent manner. This mutagenesis method is simple, fast, free of toxic chemicals, and well-suited for forward genetic screening and transgene integration.

光遗传学随机突变用组蛋白miniSOG在<em&gt;℃。线虫</em

Forward genetic screening in model organisms is the workhorse to discover functionally important genes and pathways in many biological processes. In most ...

Rearing and Double-stranded RNA-mediated Gene Knockdown in the Hide Beetle, Dermestes maculatus

Advances in genomics have raised the possibility of probing biodiversity at an unprecedented scale. However, sequence alone will not be informative without tools to study gene function. The development and sharing of detailed protocols for the establishment of new model systems in laboratories, and for tools to carry out functional studies, is thus crucial for leveraging the power of genomics. Coleoptera (beetles) are the largest clade of insects and occupy virtually all types of habitats on the planet. In addition to providing ideal models for fundamental research, studies of beetles can have impacts on pest control as they are often pests of households, agriculture, and food industries. Detailed protocols for rearing and maintenance of D. maculatus laboratory colonies and for carrying out dsRNA-mediated interference in D. maculatus are presented. Both embryonic and parental RNAi procedures-including apparatus set up, preparation, injection, and post-injection recovery-are described. Methods are also presented for analyzing embryonic phenotypes, including viability, patterning defects in hatched larvae, and cuticle preparations for unhatched larvae. These assays, together with in situ hybridization and immunostaining for molecular markers, make D. maculatus an accessible model system for basic and applied research. They further provide useful information for establishing procedures in other emerging insect model systems.

Rearing and Double-stranded RNA-mediated Gene Knockdown in the Hide Beetle, Dermestes maculatus

Here, we present protocols for rearing an intermediate-germ beetle, Dermestes maculatus (D. maculatus) in the lab. We also share protocols for embryonic and parental RNAi and methods for analyzing embryonic phenotypes to study gene function in this species.

饲养并在隐藏甲壳虫双链RNA介导的基因敲除，<em&gt;白腹皮蠹</em

Advances in genomics have raised the possibility of probing biodiversity at an unprecedented scale. However, sequence alone will not be informative ...

Microinjection of CRISPR/Cas9 Protein into Channel Catfish, Ictalurus punctatus, Embryos for Gene Editing

The complete genome of the channel catfish, Ictalurus punctatus, has been sequenced, leading to greater opportunities for studying channel catfish gene function. Gene knockout has been used to study these gene functions in vivo. The clustered regularly interspaced short palindromic repeats/CRISPR associated protein 9 (CRISPR/Cas9) system is a powerful tool used to edit genomic DNA sequences to alter gene function. While the traditional approach has been to introduce CRISPR/Cas9 mRNA into the single cell embryos through microinjection, this can be a slow and inefficient process in catfish. Here, a detailed protocol for microinjection of channel catfish embryos with CRISPR/Cas9 protein is described. Briefly, eggs and sperm were collected and then artificial fertilization performed. Fertilized eggs were transferred to a Petri dish containing Holtfreter's solution. Injection volume was calibrated and then guide RNAs/Cas9 targeting the toll/interleukin 1 receptor domain-containing adapter molecule (TICAM 1) gene and rhamnose binding lectin (RBL) gene were microinjected into the yolk of one-cell embryos. The gene knockout was successful as indels were confirmed by DNA sequencing. The predicted protein sequence alterations due to these mutations included frameshift and truncated protein due to premature stop codons.

Microinjection of CRISPR/Cas9 Protein into Channel Catfish, Ictalurus punctatus, Embryos for Gene Editing

A simple and efficient microinjection protocol for gene editing in channel catfish embryos using the CRISPR/Cas9 system is presented. In this protocol, guide RNAs and Cas9 protein were microinjected into the yolk of one-cell embryos. This protocol has been validated by knocking out two channel catfish immune-related genes.

CRISPR/Cas9 蛋白注射到沟鲶、鮰松毛虫、基因编辑的胚胎

The complete genome of the channel catfish, Ictalurus punctatus, has been sequenced, leading to greater opportunities for studying channel catfish gene ...

Embryo Microinjection and Transplantation Technique for Nasonia vitripennis Genome Manipulation

The jewel wasp Nasonia vitripennis has emerged as an effective model system for the study of processes including sex determination, haplo-diploid sex determination, venom synthesis, and host-symbiont interactions, among others. A major limitation of working with this organism is the lack of effective protocols to perform directed genome modifications. An important part of genome modification is delivery of editing reagents, including CRISPR/Cas9 molecules, into embryos through microinjection. While microinjection is well established in many model organisms, this technique is particularly challenging to perform in N. vitripennis primarily due to its small embryo size, and the fact that embryonic development occurs entirely within a parasitized blowfly pupa. The following procedure overcomes these significant challenges while demonstrating a streamlined, visual procedure for effectively removing wasp embryos from parasitized host pupae, microinjecting them, and carefully transplanting them back into the host for continuation and completion of development. This protocol will strongly enhance the capability of research groups to perform advanced genome modifications in this organism.

Embryo Microinjection and Transplantation Technique for Nasonia vitripennis Genome Manipulation

Microinjection of Nasonia vitripennis embryos is an essential method for generating heritable genome modifications. Described here is a detailed procedure for microinjection and transplantation of Nasonia vitripennis embryos, which will greatly facilitate future genome manipulation in this organism.

金小蜂金小蜂基因组操作的胚胎显微注射和移植技术

The jewel wasp Nasonia vitripennis has emerged as an effective model system for the study of processes including sex determination, haplo-diploid sex ...

CRISPR-Mediated Reorganization of Chromatin Loop Structure

Recent studies have clearly shown that long-range, three-dimensional chromatin looping interactions play a significant role in the regulation of gene expression, but whether looping is responsible for or a result of alterations in gene expression is still unknown. Until recently, how chromatin looping affects the regulation of gene activity and cellular function has been relatively ambiguous, and limitations in existing methods to manipulate these structures prevented in-depth exploration of these interactions. To resolve this uncertainty, we engineered a method for selective and reversible chromatin loop re-organization using CRISPR-dCas9 (CLOuD9). The dynamism of the CLOuD9 system has been demonstrated by successful localization of CLOuD9 constructs to target genomic loci to modulate local chromatin conformation. Importantly, the ability to reverse the induced contact and restore the endogenous chromatin conformation has also been confirmed. Modulation of gene expression with this method establishes the capacity to regulate cellular gene expression and underscores the great potential for applications of this technology in creating stable de novo chromatin loops that markedly affect gene expression in the contexts of cancer and development.

Chromatin looping plays a significant role in gene regulation; however, there have been no technological advances that allow for selective and reversible modification of chromatin loops. Here we describe a powerful system for chromatin loop re-organization using CRISPR-dCas9 (CLOuD9), demonstrated to selectively and reversibly modulate gene expression at targeted loci.

CRISPR 的染色质环结构重组

Recent studies have clearly shown that long-range, three-dimensional chromatin looping interactions play a significant role in the regulation of gene ...

Characterizing Histone Post-translational Modification Alterations in Yeast Neurodegenerative Proteinopathy Models

Neurodegenerative diseases, such as amyotrophic lateral sclerosis (ALS) and Parkinson&#39;s disease (PD), cause the loss of hundreds of thousands of lives each year. Effective treatment options able to halt disease progression are lacking. Despite the extensive sequencing efforts in large patient populations, the majority of ALS and PD cases remain unexplained by genetic mutations alone. Epigenetics mechanisms, such as the post-translational modification of histone proteins, may be involved in neurodegenerative disease etiology and progression and lead to new targets for pharmaceutical intervention. Mammalian in vivo and in vitro models of ALS and PD are costly and often require prolonged and laborious experimental protocols. Here, we outline a practical, fast, and cost-effective approach to determining genome-wide alterations in histone modification levels using Saccharomyces cerevisiae as a model system. This protocol allows for comprehensive investigations into epigenetic changes connected to neurodegenerative proteinopathies that corroborate previous findings in different model systems while significantly expanding our knowledge of the neurodegenerative disease epigenome.

This protocol outlines experimental procedures to characterize genome-wide changes in the levels of histone post-translational modifications (PTM) occurring in connection with the overexpression of proteins associated with ALS and Parkinson&#39;s disease in Saccharomyces cerevisiae models. After SDS-PAGE separation, individual histone PTM levels are detected with modification-specific antibodies via Western blotting.

酵母神经退行性蛋白病模型中组蛋白后转化修饰改变的表征

Neurodegenerative diseases, such as amyotrophic lateral sclerosis (ALS) and Parkinson&#39;s disease (PD), cause the loss of hundreds of thousands of lives ...

Determining the Egg Fertilization Rate of Bemisia tabaci Using a Cytogenetic Technique

A few species of sap-sucking whiteflies are some of the most damaging terrestrial pests worldwide because of the crop damage they inflict and plant viruses they vector. Despite numerous studies of the biology of these species in different environments, a key life history parameter, offspring sex ratios, has received little attention, yet is important for predicting population dynamics. The primary sex ratio (sex ratio at oviposition) of Bemisia tabaci has never been reported but can be found by determining the egg fertilization rate of this haplodiploid insect. The technique involves the dechorionation of eggs with bleach, a series of fixation steps, and the application of the general DNA fluorescent stain, DAPI (4&#8242;,6-diamidino-2-phenylindole, a DNA-binding fluorescent dye), to bind to female and male pronuclei. Here, we present the technique, and an example of its application, to test whether an endosymbiotic bacterium, Rickettsia sp. nr. bellii, influenced the primary sex ratio of B. tabaci. This method may assist in population studies of whiteflies, or in determining if sex allocation exists with certain environmental stimuli.

Determining the Egg Fertilization Rate of Bemisia tabaci Using a Cytogenetic Technique

We present a simple cytogenetic technique using 4&#8242;,6-diamidino-2-phenylindole (DAPI) to determine the fertilization rate and primary sex ratio of the haplodiploid invasive pest Bemisia tabaci.

用细胞遗传学技术测定芽孢杆菌的受精率

A few species of sap-sucking whiteflies are some of the most damaging terrestrial pests worldwide because of the crop damage they inflict and plant ...

Investigation of the Transcriptional Role of a RUNX1 Intronic Silencer by CRISPR/Cas9 Ribonucleoprotein in Acute Myeloid Leukemia Cells

The bulk of the human genome (~98%) is comprised of non-coding sequences. Cis-regulatory elements (CREs) are non-coding DNA sequences that contain binding sites for transcriptional regulators to modulate gene expression. Alterations of CREs have been implicated in various diseases including cancer. While promoters and enhancers have been the primary CREs for studying gene regulation, very little is known about the role of silencer, which is another type of CRE that mediates gene repression. Originally identified as an adaptive immunity system in prokaryotes, CRISPR/Cas9 has been exploited to be a powerful tool for eukaryotic genome editing. Here, we present the use of this technique to delete an intronic silencer in the human RUNX1 gene and investigate the impacts on gene expression in OCI-AML3 leukemic cells. Our approach relies on electroporation-mediated delivery of two preassembled Cas9/guide RNA (gRNA) ribonucleoprotein (RNP) complexes to create two double-strand breaks (DSBs) that flank the silencer. Deletions can be readily screened by fragment analysis. Expression analyses of different mRNAs transcribed from alternative promoters help evaluate promoter-dependent effects. This strategy can be used to study other CREs and is particularly suitable for hematopoietic cells, which are often difficult to transfect with plasmid-based methods. The use of a plasmid- and virus-free strategy allows simple and fast assessments of gene regulatory functions.

Investigation of the Transcriptional Role of a RUNX1 Intronic Silencer by CRISPR/Cas9 Ribonucleoprotein in Acute Myeloid Leukemia Cells

Direct delivery of preassembled Cas9/guide RNA ribonucleoprotein complexes is a fast and efficient means for genome editing in hematopoietic cells. Here, we utilize this approach to delete a RUNX1 intronic silencer and examine the transcriptional responses in OCI-AML3 leukemic cells.

CRISPR/Cas9核糖核酸蛋白在急性骨髓性白血病细胞中的RUNX1电子消光器的转录作用研究

The bulk of the human genome (~98%) is comprised of non-coding sequences. Cis-regulatory elements (CREs) are non-coding DNA sequences that contain binding ...