Name: ploidy manipulation of zebrafish embryos with heat shock 2 treatment
Uploaded: 2016-12-16T13:00:00
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?...

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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">
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	</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, ...

Research

JoVE Journal

Developmental Biology

Mechanical Vessel Injury in Zebrafish Embryos

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

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

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

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

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

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

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

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

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

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

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

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. ...