Name: high throughput microinjections of sea urchin zygotes
Uploaded: 2014-01-21T21:00:00
Description: Watch this Scientific Journal Video about High Throughput Microinjections of Sea Urchin Zygotes at JoVE.com

我们感谢圣地亚哥苏亚雷斯的手稿和贝蒂Cowgill援助在摄影的批判性阅读。我们也感谢匿名审稿人的批评性意见。这项工作是由特拉华大学的研究基金的支持。

1。硫酸鱼精蛋白的制备（PS）包被的培养皿<ol><li>通过在50ml锥形管中加入0.5 G PS的50毫升去离子蒸馏水（双蒸2 O）制备硫酸鱼精蛋白（PS）的1％溶液。摇匀在一条长凳上振动筛在室温下1-2小时高速，确保PS完全溶解。此溶液可以储存在4℃下至少3个月（确保在每次使用前，以完全溶解的凝胶样沉淀物）3。 </li><li>取为60毫米×15毫米的聚苯乙烯培养皿套筒，奠定了两个盖子和底部的?...

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G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Offerings+from+an+Urchin.">Offerings from an Urchin.</a> Dev. Biol. 358, 285-294 (2011).</li><li>McClay, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evolutionary+crossroads+in+developmental+biology:+sea+urchins.">Evolutionary crossroads in developmental biology: sea urchins.</a> Development. 138, 2639-2648 (2011).</li><li>Sodergren, E., 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=Research+article+-+The+genome+of+the+sea+urchin+Strongylocentrotus+purpuratus.">Research article - The genome of the sea urchin Strongylocentrotus purpuratus.</a> Science. 314, 941-952 (2006).</li><li>Roux, M. 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M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cyclin+B+synthesis+is+required+for+sea+urchin+oocyte+maturation.">Cyclin B synthesis is required for sea urchin oocyte maturation.</a> Dev. Biol. 256, 258-275 (2003).</li><li>Wong, J. L., Wessel, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extracellular+matrix+modifications+at+fertilization:+regulation+of+dityrosine+crosslinking+by+transamidation.">Extracellular matrix modifications at fertilization: regulation of dityrosine crosslinking by transamidation.</a> Development. 136, 1835-1847 (2009).</li><li>Shuster, C. B., Burgess, D. 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显微注射法是一种强大的技术用于递送各种治疗方法，如DNA，mRNA表达，重组蛋白，功能丧失和增益的功能的试剂，染料和它们的组合成蛋，胚胎，和各种生物1-7细胞。然而，设计一个显微注射实验的时候几方面的考虑，应牢记。 这是极为重要的是要考虑的递送治疗和注射量的溶解性。如果显微注射液趋向于形成即使是轻微的沉淀，在质注射针将被堵塞。一般来说，?...

高效的和可重复的治疗分娩是为研究人员的主要方法上的挑战之一。几种方法已经被建立以瞬时传送处理溶液进入卵，胚胎，和细胞。这些方法包括电穿孔（基于使用短的电脉冲的产生瞬态毛孔中的膜）1,2，脂质转染（输送通过含有治疗的脂质体与细胞膜的融合体）1，微粒轰击1（沉淀DNA上的微米这随后被用来穿透细胞在高流速大小的金属颗粒），和转导（病毒被用作转基因的递送媒介物）。此刻，显微注射是保存提供任何溶液与100％的效率以最小的试剂的优点的唯一方法。此外，单一的注射液可组成一个处理复杂的鸡尾酒。这一技术已被用于成功LY微导的卵子和胚胎从众多种类，例如海胆3,4，斑马鱼5，鼠标器6，青蛙7，和牛8以及单个细胞在组织培养9。单个卵裂球在注射后的发育阶段也已经进行了10-12。 显微注射的当前方法是基于最初由平本10所述的压力注入法，但是，大的进展已经取得了这一过程的优化。优良的显微注射技术已在别处11描述了，这里我们介绍的是目前用于微导海胆（ 紫色球海胆 ）新受精卵的具体方法之一。一个多世纪以来，海胆是一个有价值的实验模型15,16。海胆是进化密切相关的脊索动物（包括我们），并分析它们的基因组发现，它们包含所有主要的基因家族的人17。他们生产了大量的同步开发，可以很容易地操纵胚胎透明的。利用海胆作为模式生物，海胆社会对我们的受精过程的理解18-21，细胞生物学过程22-24和基因调控网络（GRNS）25-28贡献。 显微注射到海胆受精卵需要几个步骤。首先，鸡蛋需要注射之前（在下面描述），固定。显微注射的菜肴都涂有硫酸鱼精蛋白（PS），它创建了一个带正电荷的表面到带负电荷的蛋能坚持3。将卵孵育在酸性海水（pH值5.15）10分钟dejellied，随后在天然海水或人工海水（pH8.0）中洗涤两次。该dejellied鸡蛋小心划在一条直线上在1mM的3 -氨基三唑（3-AT），这是需要抑制是从卵的皮质颗粒分泌为受精29的结果ovoperoxidase的活性存在PS涂 ​​覆盘的中间。这个步骤是重要的，以防止受精信封的硬化，并促进微注射针条目。作为替代，以1mM的3-AT，10毫对氨基苯甲酸（PABA），都可以使用。注射溶液装入用专门的微负载枪头微注射针以及安装在连接到显微操纵器和压力单元的保持器（ 图1）。每根针可以用于微导合子个体中在不同的日子多个实验。可10-15分钟进行显微注射​​，直至受精卵变硬。受精卵然后用海水和培养在15℃下当到达胚胎孵化囊胚期，它们会释放孵化酶，消化外汇储备的组成部分tilization信封30，让他们自然地从PS-涂层盘分离。如果需要的话，将胚胎可以轻轻地从用口吸移管或巴斯德吸管海水轻轻地吹入胚胎培养皿分离。所描述的方法使得能够在一个单一的盘100-400新近受精的卵有效和可靠的显微注射，用于下游分析提供了一种高通量的方法。

<table border="0" cellpadding="0" cellspacing="0" style="width:793px;" width="794">
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	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Glass pasteur pipettes</td>
			<td style="width:200px;">VWR</td>
			<td style="width:211px;">14673-043</td>
			<td style="width:167px;"> </td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Inverted microscope Axiovert 40C</td>
			<td style="width:200px;">Zeiss</td>
			<td style="width:211px;">4109431007990000</td>
			<td>Injection microscope</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Microloader tips</td>
			<td style="width:200px;">Eppendorf</td>
			<td style="width:211px;">5242 956.003</td>
			<td style="width:167px;">Load injection solution</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Nylon filter mesh 80 &#956;m</td>
			<td style="width:200px;">Amazon.com</td>
			<td style="width:211px;">03-80-37</td>
			<td style="width:167px;">Filter eggs to get rid of debris</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">P20 or P200 Aerosol Barrier Pipette Tips</td>
			<td style="width:200px;">Fisher Scientific</td>
			<td style="width:211px;">02707432 or 02707430</td>
			<td style="width:167px;">Part of a mouth pipette</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Parafilm</td>
			<td style="width:200px;">Fisher Scientific</td>
			<td style="width:211px;">13 374 12</td>
			<td style="width:167px;">Part of a mouth pipette</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Polyethylene tubing</td>
			<td style="width:200px;">Intramedic</td>
			<td style="width:211px;">PE-160</td>
			<td style="width:167px;">Part of a mouth pipette</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Protamine sulfate</td>
			<td style="width:200px;">MP Biomedicals, LLC</td>
			<td style="width:211px;">194729</td>
			<td style="width:167px;">Attach dejellied eggs to injection dishes</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Sea urchins S. purpuratus</td>
			<td>Pt. Loma Marine Invertebrate Lab</td>
			<td style="width:211px;">N/A</td>
			<td style="width:167px;"> </td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Sea water</td>
			<td style="width:200px;">any pet store</td>
			<td style="width:211px;">Instant Ocean</td>
			<td style="width:167px;"> </td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Sterile 60 mm x 15 mm Polystyrene Petri Dish</td>
			<td style="width:200px;">Fisher Scientific</td>
			<td style="width:211px;">0875713A</td>
			<td style="width:167px;">Injection dishes</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Three-Axis Coarse Positioning Micromanipulator MMN-1</td>
			<td style="width:200px;">Narishige</td>
			<td style="width:211px;">9124</td>
			<td>Manipulate injection needle</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:216px;">Three-Axis Joystick Type Oil Hydraulic Fine Micromanipulator MMO-202ND</td>
			<td style="width:200px;">Narishige</td>
			<td style="width:211px;">9212</td>
			<td>Manipulate injection needle</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Transfer pipettes</td>
			<td style="width:200px;">Fisher Scientific</td>
			<td style="width:211px;">13-711-9AM</td>
			<td style="width:167px;"> </td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Vertical needle puller </td>
			<td style="width:200px;">Narishige</td>
			<td style="width:211px;">PC-10</td>
			<td>Pull injection needles</td>
		</tr>
	</tbody>
</table>

high throughput microinjections of sea urchin zygotes

显微注射到细胞和胚胎是用来研究广泛的生物学过程的一种常用技术。在该方法中少量的处理溶液被装入一个用于物理地注入单个固定的细胞或胚​​胎显微注射针。尽管需要对初始训练才能执行此程序用于高通量输送，显微注射提供最高的效率和可重复性交付各种各样的治疗方案（包括样品的复杂混合物）进入细胞，卵子或胚胎。应用显微注射包括递送的DNA构建体，mRNA表达，重组蛋白，增益功能，以及功能的试剂的损失。荧光或比色染料被加入到注射溶液，以使有效地提供实时可视化，以及提供可靠的递送溶液的量的归一化的工具。所描述的方法，使产生100-400海胆Ž显微注射在10-15分钟ygotes。

GFP和的mCherry记者构造进行体外转录和显微注射到新的受精卵。胚培养在15℃下放置24小时（直到囊胚期），并使用蔡司观察Z1显微镜成像。注射记者构造并没有导致任何发育缺陷（ 图6）。用于荧光信号的量化，图像采集是在低放大倍数（100X）进行，以最大程度地捕获荧光像素（ 图6D-F）。荧光信号使用的AxioVision 4.8.2.0定量。供的荧光信号中的100-200囊胚人口的强度?...

Watch this Scientific Journal Video about High Throughput Microinjections of Sea Urchin Zygotes at JoVE.com

High Throughput Microinjections of Sea Urchin Zygotes

显微注射是用于递送DNA构建体，mRNA表达，吗啉代反义寡核苷酸或其它处理成卵，胚胎，以及不同物种的细胞中的常见技术。

海胆受精卵的高通量显微注射

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

Research

JoVE Journal

Biology

Intravenous Microinjections of Zebrafish Larvae to Study Acute Kidney Injury

斑马鱼幼虫的研究急性肾损伤的静脉Microinjections

In this video article we describe a zebrafish model of AKI using gentamicin as the nephrotoxicant.  The technique consists of intravenous microinjections ...

科研

生物学

High-throughput Yeast Plasmid Overexpression Screen

高通量酵母质粒过表达屏幕

The budding yeast, Saccharomyces cerevisiae, is a powerful model system for defining fundamental mechanisms of many important cellular processes, ...

High-throughput Saccharification Assay for Lignocellulosic Materials

木质纤维素材料的高通量检测糖化

Polysaccharides that make up plant lignocellulosic biomass can be broken down to produce a range of sugars that subsequently can be 
used in establishing ...

High-throughput Purification of Affinity-tagged Recombinant Proteins

高通量的亲和标签的重组蛋白的纯化

X-ray crystallography is the method of choice for obtaining a detailed view of the structure of proteins. Such studies need to be complemented by further ...

Flow Cytometry-based Purification of S. cerevisiae Zygotes

Flow Cytometry-based Purification of S. cerevisiae Zygotes

流式细胞仪分离纯化<em&gt; S.酵母</em&gt;合子

Zygotes are essential intermediates between haploid and diploid states in the life cycle of many organisms, including yeast (Figure 1) 1. S. cerevisiae ...

High-throughput Titration of Luciferase-expressing Recombinant Viruses

萤光素酶表达的重组病毒的高通量滴定

Standard plaque assays to determine infectious viral titers can be time consuming, are not amenable to a high volume of samples, and cannot be done with ...

A Method for Microinjection of Patiria minata Zygotes

A Method for Microinjection of Patiria minata Zygotes

用于显微注射的方法<em&gt; Patiria minata</em&gt;合子

Echinoderms have long been a favorite model system for studies of reproduction and development, and more recently for the study of gene regulation and ...

Recovery of Adult Zebrafish Hearts for High-throughput Applications

恢复成年斑马鱼的心脏的高通量应用

Use of the zebrafish model system for studying development, regeneration, and disease is expanding toward use of adult hearts for cell dissociation and ...

High-throughput Screening for Chemical Modulators of Post-transcriptionally Regulated Genes

高通量筛选转录后调控基因的化学调节剂

Both transcriptional and post-transcriptional regulation have a profound impact on genes expression. However, commonly adopted cell-based screening assays ...

High Throughput Danio Rerio Energy Expenditure Assay

High Throughput Danio Rerio Energy Expenditure Assay

高通量<em&gt;斑马鱼</em&gt;能量消耗分析

Zebrafish are an important model organism with inherent advantages that have the potential to make zebrafish a widely applied model for the study of ...