Name: expression of fluorescent proteins in branchiostoma lanceolatum by mrna injection into unfertilized oocytes
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Description: Watch this Scientific Journal Video about Expression of Fluorescent Proteins in Branchiostoma lanceolatum by mRNA Injection into Unfertilized Oocytes at JoVE.com

笔者想承认的“Animalerie中央去伊维特河畔吉夫”的支持畜牧业。这项工作是由ANR（ANR-09-BLAN-0262-02和ANR-11-JSV2-002-01）迈克尔·舒伯特支持基金，由欧盟FP6授予“Embryomics”，并通过了ANR授予“ANR- 10 BLAN-121801开发进程“让 - 弗朗索瓦·尼古拉和纳丁Peyriéras。若昂·伊曼纽尔卡瓦略通过FCT博士奖学金资助（SFRH / BD /二千零一十二分之八万六千八百七十八）。 请求对这里所描述的载体，可以直接给作者。

1.准备仪器及试剂<ol style="margin-left: 40px;"><li>转让巴斯德移液器<ol><li>通过拉动上述火焰以不同的速度230毫米长巴斯德移液器产生一系列转印巴斯德移液器具有不同尖端的直径。确保锥度为尽可能长的愿望光滑细腻的控制。 </li><li>用金刚石划线，划伤沿线吸管垂直于吸液管的长度。用双手拉平行的吸管其长度产生钝剪。快速火焰抛光无密封尖端的吸管。 </li><li>而变化的卵母细胞/胚胎的阶段的尖?...

<ol>
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在这篇文章中，我们提出，第一次，详细的和可重复的协议，用于注射的B.卵披针叶 ，其中，经过B. floridae 13,14和B.文昌鱼 15，因此第三文昌鱼物种，其中这样的技术已被描述。重要的是，这里描述的协议也包括载体系统适合于荧光蛋白的B中生产的描述从体外产生的mRNA注入的披 （以下描述）。总之，这些新的工具允许在文昌?...

在开发过程中，单个细胞产生了一个完整的有机体中高度复杂的过程，涉及到两个细胞分裂和运动。为了更好地了解的细胞行为的动力学所依据的生物学原理，发育生物学家已经开始使用基于荧光的体内成像技术。细胞的特定区室，如细 ​​胞膜，可以通过处理来用荧光染料标记，通过组织穿透1的缺乏特异性和阻碍的方法，或由具体引入的编码荧光蛋白2外源性的mRNA胚胎。不同的技术可用于有效递送外源性化合物，如的mRNA。这些包括，但不限于，微注射，电穿孔，轰击与微粒，脂质转染和转导3,4。虽然所有这些方法可以用于引入外源性化合物成发育中的胚胎，只有微量注射允许预定义的和精确的批量应用到每个单元3。显微注射技术已被描述为所有主要发育模型系统4（ 例如 ，果蝇，线虫，斑马鱼，蛙，小鼠），以及对于某些替代模式4中 ，其中包括那些用于旨在了解发育机制的演化比较研究（ 例如 ，海葵，蠕虫环节动物，海胆，海鞘囊中，文昌鱼文昌鱼）。 Cephalochordates，连同被囊动物和脊椎动物建立脊索动物门，是特别适合的模型来研究脊索动物的演变和脊椎动物从脊椎动物的祖先5-8的多样化。在文昌鱼谱系很早脊索动物进化过程中分道扬镳;和现存cephalochordates，其被细分为三个属（ 麸chiostoma，Asymmetron和Epigonichthys），类似于脊椎动物无论是在整体解剖和基因组架构5-8的条款。已经描述到目前为止的约30种cephalochordates的五个可供胚胎和发育研究6,9：Asymmetron lucayanum（巴哈马文昌鱼）， 文昌鱼floridae（佛罗里达文昌鱼）， 文昌鱼披 （欧洲文昌鱼）， 文昌鱼 （中国的文昌鱼） 文昌鱼与日本血吸虫 （日本文昌鱼）。这三个品种的成熟的成年人（B.披针叶，B.文昌鱼和B.血吸虫 ）可诱导产卵按需在繁殖季节10,11。此外，至少对于B.披 ，高效产卵也可诱发在人造海水12，从而使这个特定文昌鱼物种laborator访问IES没有获得天然海水。的结合，在二披针叶 ，一个方便，可靠地获得胚胎具有高效的交付方法，如显微注射，至今在文昌鱼开发（在B. floridae和B.文昌鱼 ）13-15，只交付技术将使的发展新套件的操控技术，包括血统tracing-和动态细胞行为为基础的方法。 一种协议，它的mRNA的显微注射高效表达的B.荧光蛋白披针叶胚胎，因此开发。此外，提供一个基本的工具包B的实时成像披胚胎，载体系统被开发，使膜相关和荧光蛋白的细胞核表达。用于膜靶向，增强型绿色荧光蛋白（EGFP）融合到mCherry和EGFP的人的HRAS CAAX盒和核定位是由融合到斑马鱼组蛋白2B（H2B）外显子（ 图1中 ，补充文件1）中获得。此外，其目标是优化蛋白质的翻译，所述构建体含有Kozak序列和密码子已被修改，并适于使用在二披针叶 。两者合计，这里介绍的注射方法和表达载体将作为新的实验方法为cephalochordates产生的基础，特别是分析采用最新的基于荧光的体内成像技术。

<table border="0" cellpadding="0" cellspacing="0" width="1009">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">Name of Material/ Equipment</td>
			<td style="width:156px;">Company</td>
			<td style="width:143px;">Catalog Number</td>
			<td style="width:413px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">Consumables</td>
			<td style="width:156px;"></td>
			<td style="width:143px;"></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">35 mm Petri dishes</td>
			<td>Falcon</td>
			<td>353001</td>
			<td>culture-treated</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">Filtration unit (Stericup 1L)</td>
			<td style="width:156px;">Fisher</td>
			<td style="width:143px;">W21719</td>
			<td>0.22 micron filtration</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">Spin-X tubes</td>
			<td style="width:156px;">Costar</td>
			<td style="width:143px;">8160</td>
			<td>0.22 micron filtration tube</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:297px;">Needle storage jar for 1.2 mm diameter capillaries</td>
			<td style="width:156px;">WPI</td>
			<td style="width:143px;">E212</td>
			<td style="width:413px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">Pasteur Pipettes</td>
			<td style="width:156px;"></td>
			<td style="width:143px;"></td>
			<td>230 mm long</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">Aspiration tube</td>
			<td style="width:156px;">Dutscher</td>
			<td style="width:143px;">75056</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">Capillaries for injection needles</td>
			<td style="width:156px;">Sutter</td>
			<td style="width:143px;">BF 120-94-10</td>
			<td>Borosilicate glass with filament, OD 1.20 mm, ID 0.94 mm, length 10 mm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;"></td>
			<td style="width:156px;"></td>
			<td style="width:143px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">Reagents</td>
			<td style="width:156px;"></td>
			<td style="width:143px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">Low-melting agarose</td>
			<td style="width:156px;">SIGMA</td>
			<td style="width:143px;">A9414</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">Phenol Red</td>
			<td style="width:156px;">SIGMA</td>
			<td style="width:143px;">114537</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">Glycerol</td>
			<td style="width:156px;">SIGMA</td>
			<td style="width:143px;">G2025</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">Poly-L-Lysine hydrobromide</td>
			<td style="width:156px;">SIGMA</td>
			<td style="width:143px;">P9155</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">H2O Dnase, Rnase-free</td>
			<td style="width:156px;">Gibco</td>
			<td style="width:143px;">10977-035</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">mMessage mMachine SP6 Transcription kit</td>
			<td style="width:156px;">Ambion</td>
			<td style="width:143px;">AM1340</td>
			<td>mRNA synthesis kit</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">Phenol pH8</td>
			<td style="width:156px;">SIGMA</td>
			<td style="width:143px;">P4557</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">24:1 chloroform:isoamylic alcohol</td>
			<td style="width:156px;">SIGMA</td>
			<td style="width:143px;">C0549</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">5:1 phenol pH4.7:chloroform</td>
			<td style="width:156px;">SIGMA</td>
			<td style="width:143px;">P1944</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">Reef Crystal salts (200 kg)</td>
			<td style="width:156px;">Europrix&#160;</td>
			<td style="width:143px;"></td>
			<td>Commercial salts</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;"></td>
			<td style="width:156px;"></td>
			<td style="width:143px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">Equipment</td>
			<td style="width:156px;"></td>
			<td style="width:143px;"></td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:297px;">Fluorescent dissecting scope with 200x magnification&#160;</td>
			<td style="width:156px;">Leica</td>
			<td style="width:143px;">MZ16F</td>
			<td>25x oculars, DSR and GFP2 filters</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">Micromanipulator</td>
			<td style="width:156px;">Marzhauzer</td>
			<td style="width:143px;">M-33</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">Injector</td>
			<td style="width:156px;">Picospritzer</td>
			<td style="width:143px;">model II or III</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">Needle puller</td>
			<td style="width:156px;">Sutter</td>
			<td style="width:143px;">P97</td>
			<td>heating-filament needle puller</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:297px;">Fine forceps</td>
			<td>FINE SCIENCE TOOLS GMBH</td>
			<td>11252-30</td>
			<td>Dumont #5</td>
		</tr>
	</tbody>
</table>

expression of fluorescent proteins in branchiostoma lanceolatum by mrna injection into unfertilized oocytes

We report here a robust and efficient protocol for the expression of fluorescent proteins after mRNA injection into unfertilized oocytes of the cephalochordate amphioxus, Branchiostoma lanceolatum. We use constructs for membrane and nuclear targeted mCherry and eGFP that have been modified to accommodate amphioxus codon usage and Kozak consensus sequences. We describe the type of injection needles to be used, the immobilization protocol for the unfertilized oocytes, and the overall injection set-up. This technique generates fluorescently labeled embryos, in which the dynamics of cell behaviors during early development can be analyzed using the latest in vivo imaging strategies. The development of a microinjection technique in this amphioxus species will allow live imaging analyses of cell behaviors in the embryo as well as gene-specific manipulations, including gene overexpression and knockdown. Altogether, this protocol will further consolidate the basal chordate amphioxus as an animal model for addressing questions related to the mechanisms of embryonic development and, more importantly, to their evolution.

上文详述的协议提供了基础B的显微注射卵母细胞披针叶 ，因此引进来发展B. mRNA的披胚胎编码荧光蛋白用于体内成像。尽管该技术是肯定健壮和可靠的，使用此协议成功注射的速率保持变量（ 表1）。对于这样一个有趣的事实非常有可能的解释是卵母细胞离合器的极端变异性：不同蛋批次确实表现非常不同，当经受的注入压力。有些卵母细胞是相当有...

Watch this Scientific Journal Video about Expression of Fluorescent Proteins in Branchiostoma lanceolatum by mRNA Injection into Unfertilized Oocytes at JoVE.com

Expression of Fluorescent Proteins in Branchiostoma lanceolatum by mRNA Injection into Unfertilized Oocytes

我们在这里报告的强大和高效表达荧光蛋白的mRNA注射后进入披文昌鱼的未受精的卵母细胞。显微注射技术在此基础脊索动物的发展铺平道路，在这个新兴的模型系统深远的技术创新，包括在体内成像和基因特异的操作。

荧光蛋白的表达<em&gt;披文昌鱼</em&gt;通过基因注射到未受精的卵母细胞

We report here a robust and efficient protocol for the expression of fluorescent proteins after mRNA injection into unfertilized oocytes of the ...

Research

JoVE Journal

Developmental Biology

Expression of Fluorescent Proteins in Branchiostoma lanceolatum by mRNA Injection into Unfertilized Oocytes

Manipulation and In Vitro Maturation of Xenopus laevis Oocytes, Followed by Intracytoplasmic Sperm Injection, to Study Embryonic Development

Manipulation and In Vitro Maturation of Xenopus laevis Oocytes, Followed by Intracytoplasmic Sperm Injection, to Study Embryonic Development

操作和<em&gt;在体外</em中&gt;成熟<em&gt;非洲爪蟾</em&gt;卵母细胞，其次是胞浆内单精子注射，研究胚胎发育

Amphibian eggs have been widely used to study embryonic development. Early embryonic development is driven by maternally stored factors accumulated during ...

科研

发育生物学

Grafting of Beads into Developing Chicken Embryo Limbs to Identify Signal Transduction Pathways Affecting Gene Expression

珠嫁接到发展鸡胚四肢识别信号转导途径影响基因表达

Using chicken embryos it is possible to test directly the effects of either growth factors or specific inhibitors of signaling pathways on gene expression ...

Gene Transfer into the Chicken Auditory Organ by In Ovo Micro-electroporation

Gene Transfer into the Chicken Auditory Organ by In Ovo Micro-electroporation

基因转移到鸡听觉器官通过<em&gt;在卵</em&gt;微电

Chicken embryos are ideal model systems for studying embryonic development as manipulations of gene function can be conducted with relative ease in ovo. ...

Induction of Acute Skeletal Muscle Regeneration by Cardiotoxin Injection

通过注射心脏毒素急性骨骼肌再生的诱导

Skeletal muscle regeneration is a physiological process that occurs in adult skeletal muscles in response to injury or disease. Acute injury-induced ...

Immuno-fluorescent Labeling of Microtubules and Centrosomal Proteins in Ex Vivo Intestinal Tissue and 3D In Vitro Intestinal Organoids

Immuno-fluorescent Labeling of Microtubules and Centrosomal Proteins in Ex Vivo Intestinal Tissue and 3D In Vitro Intestinal Organoids

免疫荧光标记的微管和中心蛋白在体内肠道组织和 3D体外肠道 Organoids

The advent of 3D in vitro organoids that mimic the in vivo tissue architecture and morphogenesis has greatly advanced the ability to study key biological ...

Fast and Efficient Expression of Multiple Proteins in Avian Embryos Using mRNA Electroporation

使用mRNA电穿孔快速有效地表达Avian胚胎中的多种蛋白质

We report that mRNA electroporation permits fluorescent proteins to label cells in living quail embryos more quickly and broadly than DNA electroporation. ...

Hemogenic Reprogramming of Human Fibroblasts by Enforced Expression of Transcription Factors

通过转录因子的强制表达，人类纤维细胞的造血再编程

The cellular and molecular mechanisms underlying specification of human hematopoietic stem cells (HSCs) remain elusive. Strategies to recapitulate human ...

Fluorescent Immunolocalization of Arabinogalactan Proteins and Pectins in the Cell Wall of Plant Tissues

植物组织细胞壁中阿拉比诺格拉坦蛋白和皮克廷的荧光免疫化

Plant development involves constant adjustments of the cell wall composition and structure in response to both internal and external stimuli. Cell walls ...

Minimum Volume Vitrification of Immature Feline Oocytes

未成熟费林卵母细胞的最小体积玻璃化

In wild animals&#8217; conservation programs, gamete banking is crucial to safeguard genetic resources of valuable individuals and rare species and to ...

Separation of Follicular Cells and Oocytes in Ovarian Follicles of 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 ...