Name: ploidy manipulation of zebrafish embryos with heat shock 2 treatment
Uploaded: 2016-12-16T13:00:00.000+00:00
Duration: 11 min 19 s
Description: Watch this Scientific Journal Video about Ploidy Manipulation of Zebrafish Embryos with Heat Shock 2 Treatment at JoVE.com

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><tbody><tr><td>Zebrafish mating boxes</td><td>Aqua Schwarz</td><td>SpawningBox1</td><td></td></tr><tr><td>NaCl</td><td>Sigma</td><td>S5886</td><td></td></tr><tr><td>KCl</td><td>Sigma</td><td>P5405</td><td></td></tr><tr><td>Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;</td><td>Sigma</td><td>S3264</td><td></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>P9791</td><td></td></tr><tr><td>CaCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Sigma</td><td>C7902</td><td></td></tr><tr><td>MgSO&lt;sub&gt;4&lt;/sub&gt;-7H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Sigma</td><td>63138</td><td></td></tr><tr><td>NaHCO&lt;sub&gt;3&lt;/sub&gt;</td><td>Sigma</td><td>S5761</td><td></td></tr><tr><td>Tricaine</td><td>Western Chemical</td><td>Tricaine-D (MS 222)</td><td>FDA approved (ANADA 200-226)</td></tr><tr><td>Tris base</td><td>Sigma</td><td>77-86-1</td><td>to prepare 1 M Tris pH 9.0</td></tr><tr><td>HCl</td><td>Sigma</td><td>920-1</td><td>to prepare 1 M Tris pH 9.0</td></tr><tr><td>Fish net (fine mesh) (4-5 in)</td><td>PennPlax</td><td>(ThatFishThatPlace # 212370)</td><td>available in ThatFishThatPlace</td></tr><tr><td>Plastic spoon</td><td></td><td></td><td>available in most standard stores</td></tr><tr><td>Dissecting scissors</td><td>Fine Science Tools</td><td>14091-09</td><td></td></tr><tr><td>Dissecting forceps</td><td>Dumont</td><td>SS</td><td>available from Fine Science Tools</td></tr><tr><td>Dissecting stereoscope (with transmitted light source)</td><td>Nikon</td><td>SMZ645</td><td>or equivalent</td></tr><tr><td>Reflective light source (LED arms)</td><td>Fostec</td><td>KL1600 LED</td><td>or equivalent</td></tr><tr><td>Petri plates 10 cm diameter</td><td></td><td></td><td>any maker</td></tr><tr><td>Eppendorf tubes 1.5 ml</td><td></td><td></td><td>any maker</td></tr><tr><td>Ice bucket</td><td></td><td></td><td>any maker</td></tr><tr><td>Pipetteman P-1000</td><td></td><td></td><td>any maker</td></tr><tr><td>Pipette tips 1,000 &amp;micro;l</td><td></td><td></td><td>any maker</td></tr><tr><td>Narrow spatula</td><td>Fisher</td><td>14-374</td><td></td></tr><tr><td>Depression glass plate</td><td>Corning Inc</td><td>722085 (Fisher cat. No 13-748B)</td><td>available from Fisher Scientific</td></tr><tr><td>UV lamp</td><td>UVP</td><td>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><td>UV glasses</td><td></td><td></td><td>any maker</td></tr><tr><td>Paper towels</td><td></td><td></td><td>any maker</td></tr><tr><td>Kimwipes</td><td>Kimberly-Clark</td><td>06-666-11</td><td>available from Fisher Scientific</td></tr><tr><td>Timer stop watch</td><td></td><td></td><td>any maker</td></tr><tr><td>Wash bottle</td><td>Thermo Scientific</td><td>24020500</td><td>available from Fisher Scientific</td></tr><tr><td>Tea strainer</td><td></td><td></td><td>available in kitchen stores</td></tr><tr><td>beakers, 250 ml (2)</td><td>Corning Inc.</td><td>1000250</td><td>available from Fisher Scientific</td></tr><tr><td>water bath (2)</td><td></td><td></td><td>any maker, with accurary to 0.1 C (&lt;em&gt;e.g.&lt;/em&gt; Shel Lab H&lt;sub&gt;2&lt;/sub&gt;O Bath Series)</td></tr><tr><td>Hanks&amp;rsquo; Solution 1</td><td>see above</td><td>see above</td><td>8.0 g NaCl, 0.4 g KCl in 100 ml ddH&lt;sub&gt;2&lt;/sub&gt;O. Store at 4&amp;deg;C.</td></tr><tr><td>Hanks&amp;rsquo; Solution 2</td><td>see above</td><td>see above</td><td>0.358 g Anhydrous Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;, 0.6 g KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt; in 100 ml ddH&lt;sub&gt;2&lt;/sub&gt;O. Store at 4 &amp;deg;C.</td></tr><tr><td>Hanks&amp;rsquo; Solution 4</td><td>see above</td><td>see above</td><td>0.72 g CaCl&lt;sub&gt;2&lt;/sub&gt; in 50 ml ddH&lt;sub&gt;2&lt;/sub&gt;O. Store at 4 &amp;deg;C.</td></tr><tr><td>Hank&#039;s Premix</td><td>see above</td><td>see above</td><td>add, in the following order: 10.0 ml Solution 1;&amp;nbsp; 1.0 ml Solution 2;&amp;nbsp; 1.0 ml Solution 4;&amp;nbsp; 86.0 ml ddH&lt;sub&gt;2&lt;/sub&gt;O;&amp;nbsp; 1.0 ml Solution 5. Store at 4 &amp;deg;C</td></tr><tr><td>Hanks&amp;rsquo; Solution 6</td><td>see above</td><td>see above</td><td>0.33 g NaHCO&lt;sub&gt;3&lt;/sub&gt; in 10 ml ddH&lt;sub&gt;2&lt;/sub&gt;O. Prepare fresh the morning of the IVF procedure.</td></tr><tr><td>Hank&#039;s Solution (final solution)</td><td>see above</td><td>see above</td><td>Combine 990 &amp;mu;l of Hank&amp;rsquo;s Premix and 10 &amp;mu;l of freshly made Solution 6 (NaHCO&lt;sub&gt;3&lt;/sub&gt; 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

10.0K 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

Profiling DNA Replication Timing Using Zebrafish as an In Vivo Model System

DNA replication timing is an important cellular characteristic, exhibiting significant relationships with chromatin structure, transcription, and DNA mutation rates. Changes in replication timing occur during development and in cancer, but the role replication timing plays in development and disease is not known. Zebrafish were recently established as an in vivo model system to study replication timing. Here is detailed the protocols for using the zebrafish to determine DNA replication timing. After sorting cells from embryos and adult zebrafish, high-resolution genome-wide DNA replication timing patterns can be constructed by determining changes in DNA copy number through analysis of next generation sequencing data. The zebrafish model system allows for evaluation of the replication timing changes that occur in vivo throughout development, and can also be used to assess changes in individual cell types, disease models, or mutant lines. These methods will enable studies investigating the mechanisms and determinants of replication timing establishment and maintenance during development, the role replication timing plays in mutations and tumorigenesis, and the effects of perturbing replication timing on development and disease.

Profiling DNA Replication Timing Using Zebrafish as an In Vivo Model System

Zebrafish were recently used as an in vivo model system to study DNA replication timing during development. Here is detailed the protocols for using zebrafish embryos to profile replication timing. This protocol can be easily adapted to study replication timing in mutants, individual cell types, disease models, and other species.

使用斑马鱼作为体内模型系统分析 DNA 复制定时

DNA replication timing is an important cellular characteristic, exhibiting significant relationships with chromatin structure, transcription, and DNA ...

科研

遗传学

Making Gynogenetic Diploid Zebrafish by Early Pressure

Heterozygosity in diploid eukaryotes often makes genetic studies cumbersome. Methods that produce viable homozygous diploid offspring directly from heterozygous females allow F1 mutagenized females to be screened directly for deleterious mutations in an accelerated forward genetic screen. Streisinger et al.1,2 described methods for making gynogenetic (homozygous) diploid zebrafish by activating zebrafish eggs with ultraviolet light-inactivated sperm and preventing either the second meiotic or the first zygotic cell division using physical treatments (heat or pressure) that deploymerize microtubules. The "early pressure" (EP) method blocks the meiosis II, which occurs shortly after fertilization. The EP method produces a high percentage of viable embryos that can develop to fertile adults of either sex. The method generates embryos that are homozygous at all loci except those that were separated from their centromere by recombination during meiosis I. Homozygous mutations are detected in EP clutches at between 50% for centromeric loci and less than 1% for telomeric loci. This method is reproduced verbatim from the Zebrafish Book3.

This is a method for generating gynogenetic diploid zebrafish embryos (embryos whose only genetic contribution comes from the mother) by blocking the second meiotic division immediately after fertilization with ultraviolet light-inactivated sperm. EP embryos are not fully homozygous due to recombination during the first meiotic division, however they are homozygous at all loci that have not been separated from their centromere by recombination.

初压雌核发育二倍体的斑马鱼

Heterozygosity in diploid eukaryotes often makes genetic studies cumbersome. Methods that produce viable homozygous diploid offspring directly from ...

生物学

Visualizing the Developing Brain in Living Zebrafish using Brainbow and Time-lapse Confocal Imaging

Development of the vertebrate nervous system requires a precise coordination of complex cellular behaviors and interactions. The use of high resolution in vivo imaging techniques can provide a clear window into these processes in the living organism. For example, dividing cells and their progeny can be followed in real time as the nervous system forms. In recent years, technical advances in multicolor techniques have expanded the types of questions that can be investigated. The multicolor Brainbow approach can be used to not only distinguish among like cells, but also to color-code multiple different clones of related cells that each derive from one progenitor cell. This allows for a multiplex lineage analysis of many different clones and their behaviors simultaneously during development. Here we describe a technique for using time-lapse confocal microscopy to visualize large numbers of multicolor Brainbow-labeled cells over real time within the developing zebrafish nervous system. This is particularly useful for following cellular interactions among like cells, which are difficult to label differentially using traditional promoter-driven colors. Our approach can be used for tracking lineage relationships among multiple different clones simultaneously. The large datasets generated using this technique provide rich information that can be compared quantitatively across genetic or pharmacological manipulations. Ultimately the results generated can help to answer systematic questions about how the nervous system develops.

In vivo imaging is a powerful tool that can be used to investigate the cellular mechanisms underlying nervous system development. Here we describe a technique for using time-lapse confocal microscopy to visualize large numbers of multicolor Brainbow-labeled cells in real time within the developing zebrafish nervous system.

使用"彩虹"和"延时共聚焦成像"可视化活斑马鱼中发育中的脑

Development of the vertebrate nervous system requires a precise coordination of complex cellular behaviors and interactions. The use of high resolution in ...

发育生物学

Separation of Follicular Cells and Oocytes in Ovarian Follicles of Zebrafish

Zebrafish has become an ideal model to study the ovarian development of vertebrates. The follicle is the basic unit of the ovary, which consists of oocytes and surrounding follicular cells. It is vital to separate both follicular cells and oocytes for various research purposes such as for primary culture of follicular cells, analysis of gene expression, oocyte maturation and in vitro fertilization, etc. The conventional method uses forceps to separate both compartments, which is laborious, time consuming and has high damage to the oocyte. Here, we have established a simple method to separate both compartments using a pulled glass capillary. Under a stereomicroscope, oocytes and follicular cells can be easily separated by pipetting in a pulled fine glass capillary (the diameter depends on the follicle diameter). Compared with the conventional method, this new method has high efficiency in separating both oocytes and follicular cells and has low damage to the oocytes. More importantly, this method can be applied to early-stage follicles including at the pre-vitellogenesis stage. Thus, this simple method can be used to separate follicular cells and oocytes of zebrafish.

Here, we present a simple method for separating follicular cells and oocytes in zebrafish ovarian follicles, which will facilitate investigations of ovarian development in zebrafish.

斑马鱼卵巢卵巢中卵泡细胞和卵母细胞的分离

Zebrafish has become an ideal model to study the ovarian development of vertebrates. The follicle is the basic unit of the ovary, which consists of ...

A Simple and Effective Transplantation Device for Zebrafish Embryos

Classical embryological manipulations, such as removing cells and transplanting cells within or between embryos, are powerful techniques to study complex developmental processes. Zebrafish embryos are ideally suited for these manipulations since they are easily accessible, relatively large in size, and transparent. However, previously developed devices for cell removal and transplantation are cumbersome to use or expensive to purchase. In contrast, the transplantation device presented here is economical, easy to assemble, and simple to use. In this protocol, we first introduce the handling of the transplantation device as well as its assembly from commercially and widely available parts. We then present three applications for its use: generation of ectopic clones to study signal dispersal from localized sources, extirpation of cells to produce size-reduced embryos, and germline transplantation to generate maternal-zygotic mutants. Finally, we show that the tool can also be used for embryological manipulations in other species such as the Japanese rice fish medaka.

Embryological manipulations such as extirpation and transplantation of cells are important tools to study early development. This protocol describes a simple and effective transplantation device to perform these manipulations in zebrafish embryos.

一种简单有效的斑马鱼胚胎移植装置

Classical embryological manipulations, such as removing cells and transplanting cells within or between embryos, are powerful techniques to study complex ...

Rapid and Efficient Spatiotemporal Monitoring of Normal and Aberrant Cytosine Methylation within Intact Zebrafish Embryos

Cytosine methylation is highly conserved across vertebrate species and, as a key driver of epigenetic programming and chromatin state, plays a critical role in early embryonic development. Enzymatic modifications drive active methylation and demethylation of cytosine into 5-methylcytosine (5-mC) and subsequent oxidation of 5-mC into 5-hydroxymethylcytosine, 5-formylcytosine, and 5-carboxylcytosine. Epigenetic reprogramming is a critical period during in utero development, and maternal exposure to chemicals has the potential to reprogram the epigenome within offspring. This can potentially cause adverse outcomes such as immediate phenotypic consequences, long-term effects on adult disease susceptibility, and transgenerational effects of inherited epigenetic marks. Although bisulfite-based sequencing enables investigators to interrogate cytosine methylation at base-pair resolution, sequencing-based approaches are cost-prohibitive and, as such, preclude the ability to monitor cytosine methylation across developmental stages, multiple concentrations per chemical, and replicate embryos per treatment. Due to the ease of automated in vivo imaging, genetic manipulations, rapid ex utero development time, and husbandry during embryogenesis, zebrafish embryos continue to be used as a physiologically intact model for uncovering xenobiotic-mediated pathways that contribute to adverse outcomes during early embryonic development. Therefore, using commercially available 5-mC-specific antibodies, we describe a cost-effective strategy for rapid and efficient spatiotemporal monitoring of cytosine methylation within individual, intact zebrafish embryos by leveraging whole-mount immunohistochemistry, automated high-content imaging, and efficient data processing using programming language prior to statistical analysis. To current knowledge, this method is the first to successfully detect and quantify 5-mC levels in situ within zebrafish embryos during early development. The method enables the detection of DNA methylation within the cell mass and also has the ability to detect cytosine methylation of yolk-localized maternal mRNAs during the maternal-to-zygotic transition. Overall, this method will be useful for the rapid identification of chemicals that have the potential to disrupt cytosine methylation in situ during epigenetic reprogramming.

This paper describes a protocol for the rapid and efficient spatiotemporal monitoring of normal and aberrant cytosine methylation within intact zebrafish embryos.

快速有效地监测完整斑马鱼胚胎中正常和异常胞嘧啶甲基化的时空监测

Cytosine methylation is highly conserved across vertebrate species and, as a key driver of epigenetic programming and chromatin state, plays a critical ...

The Zebrafish Tol2 System: A Modular and Flexible Gateway-Based Transgenesis Approach

Fetal alcohol spectrum disorders (FASD) are characterized by a highly variable set of structural defects and cognitive impairments that arise due to prenatal ethanol exposure. Due to the complex pathology of FASD, animal models have proven critical to our current understanding of ethanol-induced developmental defects. Zebrafish have proven to be a powerful model to examine ethanol-induced developmental defects due to the high degree of conservation of both genetics and development between zebrafish and humans. As a model system, zebrafish possess many attributes that make them ideal for developmental studies, including large numbers of externally fertilized embryos that are genetically tractable and translucent. This allows researchers to precisely control the timing and dosage of ethanol exposure in multiple genetic contexts. One important genetic tool available in zebrafish is transgenesis. However, generating transgenic constructs and establishing transgenic lines can be complex and difficult. To address this issue, zebrafish researchers have established the transposon-based Tol2 transgenesis system. This modular system uses a multisite Gateway cloning approach for the quick assembly of complete Tol2 transposon-based transgenic constructs. Here, we describe the flexible Tol2 system toolbox and a protocol for generating transgenic constructs ready for zebrafish transgenesis and their use in ethanol studies.

This work describes a protocol for the modular Tol2 transgenesis system, a gateway-based cloning method to create and inject transgenic constructs into zebrafish embryos.

斑马鱼 Tol2 系统:一种模块化且灵活的基于网关的转基因方法

Fetal alcohol spectrum disorders (FASD) are characterized by a highly variable set of structural defects and cognitive impairments that arise due to ...

Triggering Cell Stress and Death Using Conventional UV Laser Confocal Microscopy

Using a standard confocal setup, a UV ablation method can be utilized to selectively induce cellular injury and to visualize single-cell responses and cell-cell interactions in the CNS in real-time. Previously, studying these cell-specific responses after injury often required complicated setups or the transfer of cells or animals into different, non-physiological environments, confounding immediate and short-term analysis. For example, drug-mediated ablation approaches often lack the specificity that is required to study single-cell responses and immediate cell-cell interactions. Similarly, while high-power pulsed laser ablation approaches provide very good control and tissue penetration, they require specialized equipment that can complicate real-time visualization of cellular responses. The refined UV laser ablation approach described here allows researchers to stress or kill an individual cell in a dose- and time-dependent manner using a conventional confocal microscope equipped with a 405-nm laser. The method was applied to selectively ablate a single neuron within a dense network of surrounding cells in the zebrafish spinal cord. This approach revealed a dose-dependent response of the ablated neurons, causing the fragmentation of cellular bodies and anterograde degeneration along the axon within minutes to hours. This method allows researchers to study the fate of an individual dying cell and, importantly, the instant response of cells-such as microglia and astrocytes-surrounding the ablation site.

Targeted manipulations to cause directed stress or death in individual cells have been relatively difficult to accomplish. Here, a single-cell-resolution ablation approach to selectively stress and kill individual cells in cell culture and living animals is described based on a standard confocal UV laser.

触发细胞应激和死亡使用传统的紫外线激光共聚焦显微镜

Using a standard confocal setup, a UV ablation method can be utilized to selectively induce cellular injury and to visualize single-cell responses and ...

神经科学

Production of Haploid Zebrafish Embryos by In Vitro Fertilization

The zebrafish has become a mainstream vertebrate model that is relevant for many disciplines of scientific study. Zebrafish are especially well suited for forward genetic analysis of developmental processes due to their external fertilization, embryonic size, rapid ontogeny, and optical clarity &#8211; a constellation of traits that enable the direct observation of events ranging from gastrulation to organogenesis with a basic stereomicroscope. Further, zebrafish embryos can survive for several days in the haploid state. The production of haploid embryos in vitro is a powerful tool for mutational analysis, as it enables the identification of recessive mutant alleles present in first generation (F1) female carriers following mutagenesis in the parental (P) generation. This approach eliminates the necessity to raise multiple generations (F2, F3, etc.) which involves breeding of mutant families, thus saving the researcher time along with reducing the needs for zebrafish colony space, labor, and the husbandry costs. Although zebrafish have been used to conduct forward screens for the past several decades, there has been a steady expansion of transgenic and genome editing tools. These tools now offer a plethora of ways to create nuanced assays for next generation screens that can be used to further dissect the gene regulatory networks that drive vertebrate ontogeny. Here, we describe how to prepare haploid zebrafish embryos. This protocol can be implemented for novel future haploid screens, such as in enhancer and suppressor screens, to address the mechanisms of development for a broad number of processes and tissues that form during early embryonic stages.

Production of Haploid Zebrafish Embryos by In Vitro Fertilization

The zebrafish is a powerful model system for developmental biology and human disease research due to their genetic similarity with higher vertebrates. This protocol describes a methodology to create haploid zebrafish embryos that can be utilized for forward screen strategies to identify recessive mutations in genes essential for early embryogenesis.

生产单倍体斑马鱼的胚胎通过<em&gt;体外</em&gt;施肥

The zebrafish has become a mainstream vertebrate model that is relevant for many disciplines of scientific study. Zebrafish are especially well suited for ...

Efficient PAM-Less Base Editing for Zebrafish Modeling of Human Genetic Disease with zSpRY-ABE8e

CRISPR/Cas9 technology has increased the value of zebrafish for modeling human genetic diseases, studying disease pathogenesis, and drug screening, but protospacer adjacent motif (PAM) limitations are a major obstacle to creating accurate animal models of human genetic disorders caused by single-nucleotide variants (SNVs). Until now, some SpCas9 variants with broad PAM compatibility have shown efficiency in zebrafish. The application of the optimized SpRY-mediated adenine base editor (ABE), zSpRY-ABE8e, and synthetically modified gRNA in zebrafish has enabled efficient adenine-guanine base conversion without PAM restriction. Described here is a protocol for efficient adenine base editing without PAM limitation in zebrafish using zSpRY-ABE8e. By injecting a mixture of zSpRY-ABE8e mRNA and synthetically modified gRNA into zebrafish embryos, a zebrafish disease model was constructed with a precise mutation that simulated a pathogenic site of the TSR2 ribosome maturation factor (tsr2). This method provides a valuable tool for the establishment of accurate disease models for studying disease mechanisms and treatments.

This protocol describes how to perform efficient adenine base editing without PAM limitation to construct a precise zebrafish disease model using zSpRY-ABE8e.

使用 zSpRY-ABE8e 对斑马鱼进行人类遗传病建模的高效无 PAM 碱基编辑

CRISPR/Cas9 technology has increased the value of zebrafish for modeling human genetic diseases, studying disease pathogenesis, and drug screening, but ...