This work was supported by NIH grants R21 HD068949-01 and RO1 GM065303.

麦迪逊和机构动物护理和使用委员会（IACUC）的指导方针（威斯康星大学 - - 麦迪逊担保数量A3368-01）所有的动物实验是根据威斯康辛大学进行的。 1.通过中断杂交选择用于鸡蛋收集女性注:根据IVF-协议依赖于成熟女性的卵子通过手工压9挤压。先前协议已经直接使用雌性从罐或成对交配而不进行卵释放行为的女性，但只有这些女性（约20％或更少，这取决于斑马鱼线）的一小部分在手动压力产生挤压和主管鸡蛋。在一个改进的过程，女性是预排序用于通过直接目视观察产蛋，随后配合的立即中断。这个过程是非常有效的，因为几乎所有的女性通过这个中断杂交一步产量挤压和主管鸡蛋预选在手动压力。 <ol><li>实验前的下午，设立在标准斑马鱼交配箱所需的斑马鱼品系交配对。保持男性在相同的腔室作为女性从他们还物理分离，或者通过一配合框除法或放置一个产卵插入下阳和插入内的女性。 </li><li>实验的早晨，取出物理分区，将两者的男性和女性相同产蛋插入内，以便配合开始。 </li><li>目测含接合对自然交配过程中检测鸡蛋的挤压坦克。在鸡蛋型材，男女分开的最初迹象打断滋生。从雄性分离后，保持预选雌性单独地或在同一水箱合并。取决于所需（通常为50-150个卵/女）胚胎数使用多个女性。 </li></ol> 2.准备一个精解注:体外受精 - [Relies成熟卵暴露于精子溶液。此溶液可以是未经处理的，以产生二倍体合子（其在HS2治疗成为四倍体胚胎）或UV处理过的，以产生单倍体合子（其在HS2治疗成为纯合二倍体胚）。以前精子制备协议建议使用细管，以从活雄性肛门区域收集鱼白，但是这是一个无效的过程，因为只有男性的一小部分，得到鱼白9。下面介绍的协议，而不是依赖于从解剖剪睾丸精子的准备，这将产生更可靠的结果。 <ol><li>提前作为汉克的预混溶液制备Hank氏溶液，其包含所有组分的（溶液1，2，4和5）除了碳酸氢钠溶液（溶液6）。制备方案6新鲜，并加入到预混物的实验（ 表1）的早晨。为了使Hanks溶液（最终解决方案，准备的早晨试管受精手术），结合汉克的预混料990 UL和10微升的新鲜出炉的解决方案6。 </li><li>由过度安乐死男性到三卡因在空调水的0.016％的溶液。 <ol><li>制备三卡因作为在水中的0.2％的储备溶液（缓冲到pH 7.0用1M的Tris pH 9.0）中，并保持在4℃。加入8毫升每100毫升水中三卡因原液在烧杯中，并用一个净给雄性转移到三卡因溶液。 </li><li>使用睾丸每需要汉克的解决方案相当于每公100微升体积受精离合器一名男性同等（从1毫升汉克的解决方案采集10名男性如睾丸，施肥10离合器用100微升/离合器）。 </li><li>确认通过鳃运动停止安乐死15分钟。安乐死后，从用勺子烧杯中取出的男性。与调节的水冲洗短暂的男性，并把它们在短暂的几个位置轻轻擦干纸巾。 </li></ol></li><li>要取出睾丸，用解剖剪刀或刀片第一斩首处死男性，并随着解剖scissors.Under解剖显微镜反射光源腹部纵行剪开，取出内脏解剖用钳。拉出每个睾丸群众钳，把它们含Hank液的离心管内。 注:睾丸可以被识别为每一个找到沿着体部的壁半透明的外观的两个细长结构和会聚泄殖腔附近。睾丸可以在解剖后一个培养皿表面并将它们放置在Hank溶液之前保持2-3分钟。 </li><li>由具有窄抹刀剪切睾丸和/或轻轻吹打起来并与1000微升尖微量溶液向下5-6倍睾丸，同时避免气泡形成释放精子进入溶液中。让睾丸碎片落户。 </li><li>存储在冰中，在那里它可保持其效力长达2-3小时精子溶液。如果在进行紫外线处理的精子溶液，将溶液转移到一个干净的微量离心管离开睾丸件后面。 </li></ol> 3.防晒修护注:UV处理是用于交联精子DNA以使其在胚胎无效。生产雌核发育单倍体的或纯合雌核发育二倍体胚胎时，这一步时才使用。紫外线处理精子的解决方案应该从睾丸（步骤2.4）件分开，因为大块可以从紫外线处理保护精子。 <ol><li>精子溶液转移到一个凹陷玻璃板坐在冰床的洁净，干燥的井（ 例如培养皿内冰）。最多可使用1毫升每玻璃板精解好。 </li><li>通过在UV灯下将所述凹陷玻璃板上冰床暴露精子溶液紫外线。把90秒的精解与115 V（60赫兹，0.68A）UV灯在19厘米（7.5英寸）的距离。用移液管尖的末端，轻轻的溶液UV处理期间，每30秒（使用干净的移液管尖端，每30秒）混合。 </li><li>使用干净的移液管尖端和微量，处理过的精子溶液转移到新的离心管中。直到需要为IVF（不超过从萃取2-3小时）在冰上储存。 </li></ol> 4.成熟的卵子的手动挤压注:由自然交配中断获得很容易产生麻醉及人工压力下的蛋女性。在此过程中，三卡因治疗应仔细控制，以避免过度，可能会阻止雌性的回收。 <ol><li>麻醉雌性通过曝光到三卡因溶液:转印女性用网，以三卡因在调节水溶液约2-5分钟，直到鱼停止（通过加入8毫升三卡因0.2％原液至100毫升的空调鱼水制成）鳃运动。 </li> &lt;LI&gt;当女性停止运动鳃，用勺子收集，并简要地调节水冲洗。依旧采用汤匙移动的鱼类，通过将其简单地反复在纸巾上的几个位置轻轻擦干，然后将它转移到一个干净，干燥的直径10厘米的培养皿。接近鱼在从前到后的方向勺，以避免潜在的破坏鳃鳃盖。 </li><li>使用实验室布或柔软的棉布轻轻进一步干燥臀鳍区域，以防止任何公布鸡蛋过早地被水激活。阻尼手指轻轻用水（避免它们粘到鱼鳞），放置一只手的一个手指上的女性的背面作为支持，并用另一只手的手指施加轻微的压力沿着女性的腹部直到鸡蛋成为挤出。 </li><li>使用窄刮刀从女性的身体移动的鸡蛋了。放置阴回到与调节的水进行回收的槽。 <br/&gt;注意:一旦而女性躺在她的身体的一侧卵挤出，她可翻转并重复在她的另一侧上的过程，以获得额外的蛋。为了确保女性的全面复苏，适用于腹部只有温和的手动压力，并开展从鳃移动停止的程序返回到水，在不到2分钟。 </li><li>激活（与水接触）和挤压后，同时90秒内受精（带精解加）鸡蛋（见第5节）。 </li></ol> 5. 在体外受精 注:天然杂交斑马鱼受精是外部的，依赖于交配期间同步上映，并活化蛋水和精子。体外受精模拟通过在水的存在下的蛋暴露于精子溶液这一过程。水体积以增加有效精子浓度为原来小（1毫升）中。精子结合15-20秒10 &lt;内发生/ SUP&gt;和水体积然后可以增加。绒毛膜通过限制窗口用于精子结合11,12权限解除进一步有助于离合器的紧密同步。因此，产生的胚胎在早期卵裂阶段主要表现同时细胞分裂周期。 <ol><li>加入100μl的精子溶液（相当于从一个男性睾丸的等效 - 见第2部分）挤出的蛋上的培养皿的离合器。轻轻摇动用于添加蛋之间的精子溶液到精子和卵子混合在一起时，应确保不从培养皿的表面抬起的尖端，以避免蛋损坏枪头。 </li><li>通过加入1ml胚胎培养基（E3）溶液的立即激活蛋（空调水也可作为在零件5至7为E3的一个替代可接受），并再次轻轻混匀卵子和精子通过用吸管尖端轻轻旋转。启动计时器启动相对施肥时机。 </l我&gt; <li>在1ml的体积1强积金，洪水与E3板。在继续之前，离开不受干扰，直到10-12强积金允许卵子活化，包括完整的绒毛膜扩张。 </li></ol> 6.热激处理注:在胚胎早期应用热休克抑制重复的中心粒，导致中心粒的不完全配套的后续细胞周期2时驱动纺锤体的形成。在缺乏沟形成13回合的结果没有主轴。 <ol><li>该绒毛膜扩张（10-12 MPF）后，倒在培养皿胚胎成滤茶器。使用的洗涤瓶E3，以便收集在滤茶器的任何剩余的胚胎冲洗培养皿。 </li><li>放置在一预加热的浴E3烧杯内的胚胎的滤茶器在28.5℃。预平衡烧杯和E3的水浴温度，并确保有足够的E3，这样在茶胚全部过滤器被暴露于介质。 </li><li>在22 MPF，从28.5℃水浴中取出滤茶器，简单地吸干到纸巾去除多余的水分，并将其放置在热浴类似预热E3烧杯内41.4℃。 </li><li>在24 MPF，短暂印迹后在28.5℃下转移滤茶器返回到E3中的水浴中。在29强积金，用洗瓶与E3的胚胎从滤茶器转移到10cm的培养皿。 </li></ol> 7.选择了胚胎一个周期胞质失速<ol><li>期间的时间段35-45 MPF，具有透射光源的解剖显微镜下，选择因此那些正在经历对称裂解成两细胞阶段，其中胚受精。删除未进行细胞分裂的胚胎。 </li><li>继续观察受精的胚胎，第一个细胞周期中选择了细胞的正常分裂，而秒内OND细胞周期（50-65 MPF）。 </li><li> 4细胞（"无失速"，在2:对于一个4芯胚胎2排列标准）根据下列类别（ 图2C）分类胚胎; 3细胞（"偏排档"，在异常2更小的单元:1大细胞排列）; 2细胞（"停止"，表现出的胚胎在1单细胞周期的延时:1的安排相同，2细胞胚胎的）。排序陷入僵局的胚胎到一个新的培养皿。 注:在此阶段，胚胎在2:2结构对应于在所述第二细胞周期期间既不卵裂球进行了细胞分裂失速;胚胎中的异常2较小的单元:1大细胞排列对应于那些热休克所述第二细胞周期中在一个卵裂球引起细胞周期失速而不是其他;和胚胎在一个1"停止":1排列对应于其中第二细胞周期期间都卵裂球经历了期望的细胞分裂摊位。停滞的胚胎应在以下细胞周期（65-80 MPF）恢复细胞分裂。卵裂球的布置可以是可变的，由于从以前的细胞周期的不完整的线索，以稳定该主轴2,14，但大多数胚胎将形成相对正常囊胚能经受正常发育。 </li><li>允许胚胎在培养皿培养，以每10cm板80的胚胎的限制。在24高倍视野，观察胚胎，以确定它们是否具有二倍体或纯合二倍体胚6（轴延伸（ 图3A，3C）的正常范围内）的一个正常的形态特征，在对比降低轴线延伸和单倍体的增大机身厚度特性（ 图3B）），和/或使用遗传颜料标记在36 MPF，如金或白化等这样的测定（ 图3和参见下文）2,6,9-评估二倍。 </li&gt; <li>删除似乎有一个单倍体的形态，或已发生裂解或出现其他严重缺陷，异常胚胎的任何。 注:如果需要的话，允许胚胎发展，直到后5天受精，同时继续以除去每天裂解或严重异常胚胎和加入新鲜E3刷新介质。注:生存胚胎也可以通过转移到孵化系统和标准条件下饲养鳔充气后生长的第5天。 </li></ol>

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B., Bullejos, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Coexistence+of+Y,+W,+and+Z+sex+chromosomes+in+Xenopus+tropicalis.">Coexistence of Y, W, and Z sex chromosomes in Xenopus tropicalis.</a> Proc. Natl. Acad. Sci. USA. 112, 4752-4761 (2015).</li><li>Streisinger, G., Singer, F., Walker, C., Knauber, D., Dower, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Segregation+analyses+and+gene-centromere+distances+in+zebrafish.">Segregation analyses and gene-centromere distances in zebrafish.</a> Genetics. 112, 311-319 (1986).</li><li>Dekens, M. P. S., Pelegri, F. J., Maischein, H. -. M., N&#252;sslein-Volhard, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+maternal-effect+gene+futile+cycle.is+essential+for+pronuclear+congression+and+mitotic+spindle+assembly+in+the+zebrafish+zygote.">The maternal-effect gene futile cycle.is essential for pronuclear congression and mitotic spindle assembly in the zebrafish zygote.</a> Development. 130, 3907-3916 (2003).</li><li>Pelegri, F., Mullins, M., Detrich, H. W., Westerfield, M., Zon, L. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Met. Cell Biol. Vol. 104. , 83-120 (2011).</li></ol>

The authors do not have competing financial interests.

关键步骤它是在体外受精的有效条件下工作的关键。为了保证成熟的卵子（步骤1）良好的供应，女性设立交配不应该建立在交配十字架至少5天，应该会出现妊娠。养殖期间中断，观察者能够充分监督15-30坦克天然蛋挤压的首次亮相。交配的中断应​​当第一蛋是通过自然交配释放，以允许最蛋留在女性为体外受精过程内，就会产生可能的。这些女性，目前已准备成熟的卵子人?...

该协议允许倍性在斑马鱼胚胎，诸如在从核发育单倍体（ 图1）或生产四倍体纯合雌核二倍体的产生操作。这是通过诱导对应于正好一个细胞周期（ 图2A，2B）胞质的延迟来实现的。在胞质分裂的关键一个周期的延迟是通过用热休克实现。热休克（HS）最初由Streisinger和同事描述的标准协议的周期13-15 MPF期间所涉及的温度下脉冲，第一细胞周期1期间导致一个周期细胞分裂失速。此协议的效率与热休克治疗的滑动时间窗口扫描早期细胞周期最近的改善。该扫描确定了一个热休克稍后的时间点，仍是第一细胞周期（22-24 MPF）内，即导致较高的速率EMBR的YOS与一个周期的细胞分裂失速，而在这种情况下，会影响第二细胞周期2。该第一细胞周期中的实验操作的第二单元周期期间干扰细胞分裂和导致DNA含量复制观察也有报道在其他鱼类3,4。我们将此改性协议作为热休克2（HS2 - 术语"2"反映了加热脉冲发生在比标准的HS方法稍后的时间点，而第二细胞周期过程中发生引起HS2细胞周期延迟）。这些研究表明，对于细胞分裂停滞热休克后的基础是中心粒复制的热脉冲，从而影响在下面的细胞周期纺锤体形成与沟诱导时的抑制。 HS2导致细胞周期停滞的收率接近100％，倍性复制到高4倍，比标准的HS 2速率。 胚胎机智处理哈卵裂球细胞循环过程中的热休克表现出许多有害的影响，这表明热休克影响细胞分裂2需要多个进程。另一方面，当热冲击前的细胞周期（时间周期0-30 MPF）的开始施加，它似乎有与中心粒复制特定干扰一致的效应并似乎不影响其它基本细胞过程2 。这些研究表明，之前卵裂球分裂的开始时间似乎是一个发展期适合使用热休克作为一种工具来具体操作，通过中心粒抑制倍。对中心粒复制热休克表观选择性的根本原因尚不清楚，但可能与在特定细胞类型中的热应力下观察中心体子结构，例如白细胞5的选择性降解。 胚胎去的时间同步发展最是通过在体外受精（IVF）来实现的。在二倍体胚胎受精结果在HS2诱导的单周期失速胞质四倍体成为精子未经使用。采用UV处理过的精子，它携带灭活其DNA交联，结果雌核发育单倍体胚胎6，其在HSII诱导的单周期胞质失速成为雌核发育二倍体2。因为得到的全基因组倍增的，后者雌核二倍体是在整个基因组中的每一个基因座纯合的。为简明起见，我们指的是雌核单倍体胚为"单倍体"，和纯合雌核发育二倍体胚为"纯合二倍体"。如果可行的肥沃，纯合二倍体可用于发起无菌和无致死线。通过HS2诱导直接纯合性也应容易地并入基因分析或遗传筛选中，由于纯合二倍体从女性是MUT的杂合子携带者在高和固定的（50％）比2纯合子的ations展览率。 以下协议描述的步骤来执行HS2和诱导倍体复制具有完全纯合。对于四倍体生产，精子溶液应治疗。为纯合二倍体生产，精子应通过UV处理而失活。此外，如在讨论中所述，可见色素标记也可用于促进纯合二倍体鉴定。主要在他们的光变周期7，和成人和鸡蛋开始的第一个3小时斑马鱼的伴侣是昼夜节律8敏感，所以为了获得最佳效果的试管受精手术应该在这段时间内发生。

<table border="0" cellpadding="0" cellspacing="0" width="573">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Zebrafish mating boxes</td>
			<td style="width:71px;">Aqua Schwarz</td>
			<td style="width:119px;">SpawningBox1</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">NaCl</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:119px;">S5886</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">KCl</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:119px;">P5405</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Na2HPO4</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:119px;">S3264</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">KH2PO4</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:119px;">P9791</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">CaCl2</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:119px;">C7902</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">MgSO4-7H2O</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:119px;">63138</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">NaHCO3</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:119px;">S5761</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Tricaine</td>
			<td style="width:71px;">Western Chemical</td>
			<td style="width:119px;">Tricaine-D (MS 222)</td>
			<td>FDA approved (ANADA 200-226)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Tris base</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:119px;">77-86-1</td>
			<td>to prepare 1 M Tris pH 9.0</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">HCl</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:119px;">920-1</td>
			<td>to prepare 1 M Tris pH 9.0</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Fish net (fine mesh) (4-5 in)</td>
			<td style="width:71px;">PennPlax</td>
			<td style="width:119px;">(ThatFishThatPlace # 212370)</td>
			<td>available in ThatFishThatPlace</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Plastic spoon</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td>available in most standard stores</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Dissecting scissors</td>
			<td style="width:71px;">Fine Science Tools</td>
			<td style="width:119px;">14091-09</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Dissecting forceps</td>
			<td style="width:71px;">Dumont</td>
			<td style="width:119px;">SS</td>
			<td>available from Fine Science Tools</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Dissecting stereoscope (with transmitted light source)</td>
			<td style="width:71px;">Nikon</td>
			<td style="width:119px;">SMZ645</td>
			<td>or equivalent</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Reflective light source (LED arms)</td>
			<td style="width:71px;">Fostec</td>
			<td style="width:119px;">KL1600 LED</td>
			<td>or equivalent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Petri plates 10 cm diameter</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td>any maker</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Eppendorf tubes 1.5 ml</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td>any maker</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Ice bucket</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td>any maker</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Pipetteman P-1000</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td>any maker</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Pipette tips 1000 &#181;l</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td>any maker</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Narrow spatula</td>
			<td style="width:71px;">Fisher</td>
			<td style="width:119px;">14-374</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Depression glass plate</td>
			<td style="width:71px;">Corning Inc</td>
			<td style="width:119px;">722085 (Fisher cat. No 13-748B)</td>
			<td>available from Fisher Scientific</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">UV lamp</td>
			<td style="width:71px;">UVP</td>
			<td style="width:119px;">Model XX-15 (cat No. UVP18006201)</td>
			<td>available from Fisher Scientific. Although not observed by us with this model, some UV sources have been observed to experience a decrease of intensity over time (if this is the case, see Modifications and Troubleshooting)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">UV glasses</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td>any maker</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Paper towels</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td>any maker</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Kimwipes</td>
			<td style="width:71px;">Kimberly-Clark</td>
			<td style="width:119px;">06-666-11</td>
			<td>available from Fisher Scientific</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Timer stop watch</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td>any maker</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Wash bottle</td>
			<td style="width:71px;">Thermo Scientific</td>
			<td style="width:119px;">24020500</td>
			<td>available from Fisher Scientific</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Tea strainer</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td>available in kitchen stores</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">beakers, 250 ml (2)</td>
			<td style="width:71px;">Corning Inc.</td>
			<td style="width:119px;">1000250</td>
			<td>available from Fisher Scientific</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">water bath (2)</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td>any maker, with accurary to 0.1 C (e.g. Shel Lab H2O Bath Series)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;"></td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="44">
			<td height="44" style="height:44px;">Hanks&#8217; Solution 1</td>
			<td style="width:71px;">see above</td>
			<td style="width:119px;">see above</td>
			<td>8.0g NaCl, 0.4g KCl in 100ml ddH2O. Store at 4&#176;C.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Hanks&#8217; Solution 2</td>
			<td style="width:71px;">see above</td>
			<td style="width:119px;">see above</td>
			<td>0.358g Anhydrous Na2HPO4, 0.6g KH2PO4 in 100ml ddH2O. Store at 4&#176;C.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Hanks&#8217; Solution 4</td>
			<td style="width:71px;">see above</td>
			<td style="width:119px;">see above</td>
			<td>0.72g CaCl2 in 50ml ddH2O. Store at 4&#176;C.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Hanks&#8217; Solution 5</td>
			<td style="width:71px;">see above</td>
			<td style="width:119px;">see above</td>
			<td>1.23g MgSO4 &#8729; 7H2O in 50ml ddH2O. Store at 4&#176;C.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;"></td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Hank&#39;s Premix</td>
			<td style="width:71px;">see above</td>
			<td style="width:119px;">see above</td>
			<td>add, in the following order: 10.0 ml Solution 1;&#160; 1.0 ml Solution 2;&#160; 1.0 ml Solution 4;&#160; 86.0 ml ddH2O;&#160; 1.0 ml Solution 5. Store at 4&#176;C</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;"></td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Hanks&#8217; Solution 6</td>
			<td style="width:71px;">see above</td>
			<td style="width:119px;">see above</td>
			<td>0.33g NaHCO3 in 10ml ddH2O. Prepare fresh the morning of the IVF procedure.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;"></td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Hank&#39;s Solution (final solution)</td>
			<td style="width:71px;">see above</td>
			<td style="width:119px;">see above</td>
			<td>Combine 990ul of Hank&#8217;s Premix and 10ul of freshly made Solution 6 (NaHCO3 solution)</td>
		</tr>
	</tbody>
</table>

ploidy manipulation of zebrafish embryos with heat shock 2 treatment

倍体的操纵允许有用的变换，如二倍体对四倍体或单倍体到二倍体。在斑马鱼 ，特别是因为它允许从一个单一的杂合母直接生产纯合子的纯合雌核二倍体的生成是在遗传分析是有用的。本文介绍的第一个细胞周期，热冲击2（HS2）期间，基于热脉冲重复套数修改后的协议。通过抑制中心粒复制的，此方法的第二细胞周期中产生一个精确的细胞分裂失速。精确的单周期划分摊位，耦合到未受影响DNA复制，导致了全基因组倍增。用这种方法相关的协议包括卵子和精子采集，精子的UV处理， 体外受精和热脉冲引起一个细胞周期分裂延迟和重复套数。本协议的修改后的版本可以应用以诱导其它动物物种的倍性的变化。

尽管单细胞周期胞质失速，DNA复制在这样的胚胎通常发生，导致胚胎（ 图1）的DNA的内容的复制。该Streisinger热休克协议（标准HS）期间13-15分钟后受精（MPF）中涉及到热脉冲，并在35 MPF 1,2第一胚胎细胞分裂过程中主要诱导细胞分裂逮捕，而这里介绍的方法得出，称为热休克2（HS2），周期22-24 MPF期间使用的热冲击，并在50 MPF（ 图2; 2,3,4?...

Watch this Scientific Journal Video about Ploidy Manipulation of Zebrafish Embryos with Heat Shock 2 Treatment at JoVE.com

Ploidy Manipulation of Zebrafish Embryos with Heat Shock 2 Treatment

对于套数操纵修改协议使用热休克诱导胞质一个周期的摊位在早期胚胎。这个协议是表明在斑马鱼，也可以是适用于其它物种。

斑马鱼胚胎的倍性操作与热休克治疗2

Manipulation of ploidy allows for useful transformations, such as diploids to tetraploids, or haploids to diploids. In the zebrafish Danio rerio, ...

ploidy-manipulation-of-zebrafish-embryos-with-heat-shock-2-treatment

Research

JoVE Journal

Developmental Biology

9.9K 次观看.  University of Wisconsin. 对于套数操纵修改协议使用热休克诱导胞质一个周期的摊位在早期胚胎。这个协议是表明在斑马鱼，也可以是适用于其它物种。 

视频: Ploidy Manipulation of Zebrafish Embryos with Heat Shock 2 Treatment

Mechanical Vessel Injury in Zebrafish Embryos

Zebrafish (Danio rerio) embryos have proven to be a powerful model for studying a variety of developmental and disease processes. External development and optical transparency make these embryos especially amenable to microscopy, and numerous transgenic lines that label specific cell types with fluorescent proteins are available, making the zebrafish embryo an ideal system for visualizing the interaction of vascular, hematopoietic, and other cell types during injury and repair in vivo. Forward and reverse genetics in zebrafish are well developed, and pharmacological manipulation is possible. We describe a mechanical vascular injury model using micromanipulation techniques that exploits several of these features to study responses to vascular injury including hemostasis and blood vessel repair. Using a combination of video and timelapse microscopy, we demonstrate that this method of vascular injury results in measurable and reproducible responses during hemostasis and wound repair. This method provides a system for studying vascular injury and repair in detail in a whole animal model.

This article describes a method for creating a mechanical vessel injury in zebrafish embryos. This injury model provides a platform for studying hemostasis, injury-related inflammation, and wound healing in an organism ideally suited for real-time microscopy.

在斑马鱼胚胎的机械血管损伤

Zebrafish (Danio rerio) embryos have proven to be a powerful model for studying a variety of developmental and disease processes.  External development ...

科研

发育生物学

Isolation and Characterization of Single Cells from Zebrafish Embryos

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

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

分离和斑马鱼胚胎单细胞的表征

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

Generation of Parabiotic Zebrafish Embryos by Surgical Fusion of Developing Blastulae

Surgical parabiosis of two animals of different genetic backgrounds creates a unique scenario to study cell-intrinsic versus cell-extrinsic roles for candidate genes of interest, migratory behaviors of cells, and secreted signals in distinct genetic settings. Because parabiotic animals share a common circulation, any blood or blood-borne factor from one animal will be exchanged with its partner and vice versa. Thus, cells and molecular factors derived from one genetic background can be studied in the context of a second genetic background. Parabiosis of adult mice has been &#160;used extensively to research aging, cancer, diabetes, obesity, and brain development. More recently, parabiosis of zebrafish embryos has been used to study the developmental biology of hematopoiesis. In contrast to mice, the transparent nature of zebrafish embryos permits the direct visualization of cells in the parabiotic context, making it a uniquely powerful method for investigating fundamental cellular and molecular mechanisms. The utility of this technique, however, is limited by a steep learning curve for generating the parabiotic zebrafish embryos. This protocol provides a step-by-step method on how to surgically fuse the blastulae of two zebrafish embryos of different genetic backgrounds to investigate the role of candidate genes of interest. In addition, the parabiotic zebrafish embryos are tolerant to heat shock, making temporal control of gene expression possible. This method does not require a sophisticated set-up and has broad applications for studying cell migration, fate specification, and differentiation in vivo during embryonic development.

This protocol provides step-by-step instruction on how to generate parabiotic zebrafish embryos of different genetic backgrounds. When combined with the unparalleled imaging capabilities of the zebrafish embryo, this method provides a uniquely powerful means to investigate cell-autonomous versus non-cell-autonomous functions for candidate genes of interest.

联体斑马鱼胚胎一代发展囊胚的手术融合

Surgical parabiosis of two animals of different genetic backgrounds creates a unique scenario to study cell-intrinsic versus cell-extrinsic roles for ...

Affinity Labeling Detection of Endogenous Receptors from Zebrafish Embryos

By combining the powers of Affinity Labeling and Immunoprecipitation (AFLIP), a technique for the detection of low abundance receptors in zebrafish embryos has been implemented. This technique takes advantage of the selectivity and sensitivity conferred by affinity labeling of a given receptor by its ligand with the specificity of the immunoprecipitation. We have used AFLIP to detect the type III TGF-&#946; receptor (TGFBR3), also know as betaglycan, during early zebrafish development. AFLIP was instrumental in validating the efficacy of a TGFBR3 morphant zebrafish phenotype. In the first step, embryo protein extracts are prepared and used to generate 125I-TGF-&#946;2-TGFBR3 complexes that are purified by immunoprecipitation. Later, these complexes are covalently cross-linked and revealed using SDS-PAGE separation and autoradiography detection. This technique requires the availability of a labeled ligand for, and a specific antibody against, the receptor to be detected, and shall be easily adapted to identify any growth factor or cytokine receptor that meets these requirements.

A novel technique for the detection of low abundance endogenous receptors present in zebrafish embryos is described. We have named it AFLIP because it consists of affinity labeling of the receptor by its ligand linked to immunoprecipitation.

从斑马鱼胚胎内源性受体亲和标记检测

By combining the powers of Affinity Labeling and Immunoprecipitation (AFLIP), a technique for the detection of low abundance receptors in zebrafish ...

Functional Manipulation of Maternal Gene Products Using In Vitro Oocyte Maturation in Zebrafish

Cellular events that take place during the earliest stages of animal embryonic development are driven by maternally derived gene products deposited into the developing oocyte. Because these events rely on maternal products which typically act very soon after fertilization-that preexist inside the egg, standard approaches for expression and functional reduction involving the injection of reagents into the fertilized egg are typically ineffective. Instead, such manipulations must be performed during oogenesis, prior to or during the accumulation of maternal products. This article describes in detail a protocol for the in vitro maturation of immature zebrafish oocytes and their subsequent in vitro fertilization, yielding viable embryos that survive to adulthood. This method allows the functional manipulation of maternal products during oogenesis, such as the expression of products for phenotypic rescue and tagged construct visualization, as well as the reduction of gene function through reverse-genetics agents.

Functional Manipulation of Maternal Gene Products Using In Vitro Oocyte Maturation in Zebrafish

An optimized protocol for the in vitro maturation of zebrafish oocytes used for the manipulation of maternal gene products is presented here.

产妇基因产品的官能化操作使用<em&gt;体外</em斑马鱼&gt;卵母细胞成熟

Cellular events that take place during the earliest stages of animal embryonic development are driven by maternally derived gene products deposited into ...

Biosensing Motor Neuron Membrane Potential in Live Zebrafish Embryos

The protocols described here are designed to allow researchers to study cell communication without altering the integrity of the environment in which the cells are located. Specifically, they have been developed to analyze the electrical activity of excitable cells, such as spinal neurons. In such a scenario, it is crucial to preserve the integrity of the spinal cell, but it is also important to preserve the anatomy and physiological shape of the systems involved. Indeed, the comprehension of the manner in which the nervous system-and other complex systems-works must be based on a systemic approach. For this reason, the live zebrafish embryo was chosen as a model system, and the spinal neuron membrane voltage changes were evaluated without interfering with the physiological conditions of the embryos. 
Here, an approach combining the employment of zebrafish embryos with a FRET-based biosensor is described. Zebrafish embryos are characterized by a very simplified nervous system and are particularly suited for imaging applications thanks to their transparency, allowing for the employment of fluorescence-based voltage indicators at the plasma membrane during zebrafish development. The synergy between these two components makes it possible to analyze the electrical activity of the cells in intact living organisms, without perturbing the physiological state. Finally, this non-invasive approach can co-exist with other analyses (e.g., spontaneous movement recordings, as shown here).

Protocols described here allow for the study of the electrical properties of excitable cells in the most non-invasive physiological conditions by employing zebrafish embryos in an in vivo system together with a fluorescence resonance energy transfer (FRET)-based genetically encoded voltage indicator (GEVI) selectively expressed in the cell type of interest.

生物传感运动神经元膜电位在活斑马鱼胚胎

The protocols described here are designed to allow researchers to study cell communication without altering the integrity of the environment in which the ...

Induction of Hypoxia in Living Frog and Zebrafish Embryos

Here, we introduce a novel system for hypoxia induction, which we developed to study the effects of hypoxia in aquatic organisms such as frog and zebrafish embryos. Our system comprises a chamber featuring a simple setup that is nevertheless robust to induce and maintain a specific oxygen concentration and temperature in any experimental solution of choice. The presented system is very cost-effective but highly functional, it allows induction and sustainment of hypoxia for direct experiments in vivo and for various time periods up to 48 h.
To monitor and study the effects of hypoxia, we have employed two methods - measurement of levels of hypoxia-inducible factor 1alpha (HIF-1&#945;) in whole embryos or specific tissues and determination of retinal stem cell proliferation by 5-ethynyl-2&#8242;-deoxyuridine (EdU) incorporation into the DNA. HIF-1&#945; levels can serve as a general hypoxia marker in the whole embryo or tissue of choice, here embryonic retina. EdU incorporation into the proliferating cells of embryonic retina is a specific output of hypoxia induction. Thus, we have shown that hypoxic embryonic retinal progenitors decrease proliferation within 1 h of incubation under 5% oxygen of both frog and zebrafish embryos.
Once mastered, our setup can be employed for use with small aquatic model organisms, for direct in vivo experiments, any given time period and under normal, hypoxic or hyperoxic oxygen concentration or under any other given gas mixture.

We introduce a novel hypoxic chamber system for use with aquatic organisms such as frog and zebrafish embryos. Our system is simple, robust, cost-effective and allows the induction and sustainment of hypoxia in vivo and for up to 48 h. We present 2 reproducible methods to monitor the effectiveness of hypoxia.

诱导生活青蛙和斑马鱼胚胎缺氧

Here, we introduce a novel system for hypoxia induction, which we developed to study the effects of hypoxia in aquatic organisms such as frog and ...

Application of RNAi and Heat-shock-induced Transcription Factor Expression to Reprogram Germ Cells to Neurons in C. elegans

Studying the cell biological processes during converting the identities of specific cell types provides important insights into mechanism that maintain and protect cellular identities. The conversion of germ cells into specific neurons in the nematode Caenorhabditis elegans (C. elegans) is a powerful tool for performing genetic screens in order to dissect regulatory pathways that safeguard established cell identities. Reprogramming of germ cells to a specific type of neurons termed ASE requires transgenic animals that allow broad over-expression of the Zn-finger transcription factor (TF) CHE-1. Endogenous CHE-1 is expressed exclusively in two head neurons and is required to specify the glutamatergic ASE neurons fate, which can easily be visualized by the gcy-5prom::gfp reporter. A trans gene containing the heat-shock promoter-driven che-1 gene expression construct allows broad mis-expression of CHE-1 in the entire animal upon heat-shock treatment. The combination of RNAi against the chromatin-regulating factor LIN-53 and heat-shock-induced che-1 over-expression leads to reprogramming of germ cell into ASE neuron-like cells. We describe here the specific RNAi procedure and appropriate conditions for heat-shock treatment of transgenic animals in order to successfully induce germ cell to neuron conversion.

Application of RNAi and Heat-shock-induced Transcription Factor Expression to Reprogram Germ Cells to Neurons in C. elegans

This protocol describes how to study cellular processes during cell fate conversion in Caenorhabditis elegans in vivo. Using transgenic animals, allowing heat-shock promoter-driven overexpression of the neuron fate-inducing transcription factor CHE-1 and RNAi-mediated depletion of the chromatin-regulating factor LIN-53 germ cell to neuron reprogramming can be observed in vivo.

rna 干扰和热休克诱导转录因子表达在重编程生殖细胞到神经元中的应用C. 线虫

Studying the cell biological processes during converting the identities of specific cell types provides important insights into mechanism that maintain ...

Visualization of Cellular Electrical Activity in Zebrafish Early Embryos and Tumors

Bioelectricity, endogenous electrical signaling mediated by ion channels and pumps located on the cell membrane, plays important roles in signaling processes of excitable neuronal and muscular cells and many other biological processes, such as embryonic developmental patterning. However, there is a need for in vivo electrical activity monitoring in vertebrate embryogenesis. The advances of genetically encoded fluorescent voltage indicators (GEVIs) have made it possible to provide a solution for this challenge. Here, we describe how to create a transgenic voltage indicator zebrafish using the established voltage indicator, ASAP1 (Accelerated Sensor of Action Potentials 1), as an example. The Tol2 kit and a ubiquitous zebrafish promoter, ubi, were chosen in this study. We also explain&#160;the processes of Gateway site-specific cloning, Tol2 transposon-based zebrafish transgenesis, and the imaging process for early-stage fish embryos and fish tumors using regular epifluorescent microscopes. Using this fish line, we found that there are cellular electric voltage changes during zebrafish embryogenesis, and fish larval movement. Furthermore, it was observed that in a few zebrafish malignant peripheral nerve sheath tumors, the tumor cells were generally polarized compared to the surrounding normal tissues.

Here, we show the process of creating a cellular electric voltage reporter zebrafish line to visualize embryonic development, movement, and fish tumor cells in vivo.

斑马鱼早期胚胎和肿瘤细胞电活动的可视化研究

Bioelectricity, endogenous electrical signaling mediated by ion channels and pumps located on the cell membrane, plays important roles in signaling ...

Whole Mount Immunohistochemistry in Zebrafish Embryos and Larvae

Immunohistochemistry is a widely used technique to explore protein expression and localization during both normal developmental and disease states. Although many immunohistochemistry protocols have been optimized for mammalian tissue and tissue sections, these protocols often require modification and optimization for non-mammalian model organisms. Zebrafish are increasingly used as a model system in basic, biomedical, and translational research to investigate the molecular, genetic, and cell biological mechanisms of developmental processes. Zebrafish offer many advantages as a model system but also require modified techniques for optimal protein detection. Here, we provide our protocol for whole-mount fluorescence immunohistochemistry in zebrafish embryos and larvae. This protocol additionally describes several different mounting strategies that can be employed and an overview of the advantages and disadvantages each strategy provides. We also describe modifications to this protocol to allow detection of chromogenic substrates in whole mount tissue and fluorescence detection in sectioned larval tissue. This protocol is broadly applicable to the study of many developmental stages and embryonic structures.

Here, we present a protocol for fluorescent antibody-mediated detection of proteins in whole preparations of zebrafish embryos and larvae.

斑马鱼胚胎和幼虫的整个山免疫性化学

Immunohistochemistry is a widely used technique to explore protein expression and localization during both normal developmental and disease states. ...