这项工作是由G.哈罗德和莱拉华马瑟斯奖HL和美国国立卫生研究院资助（R24 GM080527），以RESCEJ是一个NRSA博士后研究员（NIH F32GM9037222），目前由指导式研究科学家开发奖由国家支持老年研究所（K01AG04435）。我们要感谢评审的有益的意见。

1，制备质粒DNA，针头和注射的菜<ol><li>准备使用高速马克西和Midi试剂盒的质粒DNA，并收集DNA，以2.5毫克/毫升用乙醇沉淀。将DNA沉淀应溶解于无核酸酶的去离子水。存储在10-20微升等份的DNA于-20℃。 （可用矢量列表，请参阅补充表1） </li><li>用琼脂糖构建一个低谷举行的胚胎在100×15毫米的培养皿使注射的菜。 <ol><li>将一个75×50显微镜载玻片成100×15毫米的培养皿以一定的角度。倒入约50ml的2％的琼脂糖熔化水润介质24到培养皿中。或者，先倒入琼脂糖入菜，并飘起了“显微注射模具的鱼”在上面。 </li><li>经过琼脂糖凝固，取出载玻片或模具。如果使用载玻片上，切掉多余的琼脂糖用剃刀BLA德成墙。 水润凝中等入菜，并立即使用它，或它在4℃保存备用。注意：如果储存在4℃下使用之间注射菜肴可重复使用。 </li></ol></li><li>拉从高硼硅玻璃毛细管针，灯丝使用下列条件的微量拉马：加热525，拉75，速度100，时间50商店针按成泥的狭长地带在培养皿中，使他们手持平行于培养皿的底部。 </li><li>制备注射溶液，结合3微升2.5毫克/毫升的质粒DNA溶液与2微升10％酚红。涡旋该溶液，随后又旋转它10分钟，在最大速度下在微量离心以除去可能堵塞针的任何颗粒。 </li><li>在解剖显微镜下，夹针从尖端几毫米用钳子来创建一个小口。注意：在打开的适当大小有一定的灵活性荷兰国际集团，因为压力可以在步骤3.3进行调整，以适应这种变化。 </li><li>放置在培养皿，以便它保持在针与所述尖端向下的竖直位置。由液体吸取入针的后端装入大约0.5微升注射溶液的针。允许毛细管作用拉动溶液进针的尖端。 </li></ol> 2，准备胚胎注射<ol><li>每天扫描息肉产蛋水润 AEP的菌种。收集这些息肉和将它们搁置在一个单独的培养皿。白天观察这些息肉每隔几个小时监测卵形成的进展。当鸡蛋突破外胚层和坐在缩回 ​​外胚层细胞环，他们已经准备好注射25。把这些息肉有几个水润有睾丸注射，以便受精前至少1小时的菜。 水润&lt;/ em&gt;的雄性将精子释放没有任何特殊的操作。 </li><li>如果女性被发现在AEP菌落与完全成形的鸡蛋，这是大约400微米的直径25，或1至8细胞期胚胎中，也收集这些用于注射。立即注入已经开始切割胚胎。注意：这是不可能的，告诉完全成形的鸡蛋和单细胞受精卵的区别，因而这些应孵育注射前男性。 </li><li>注射前，除去大部分的亲组织的上方和下方用手术刀以＃15刀片的胚胎。只留下胚胎用一小片体柱的附着，所使用，如果必要的操纵胚胎用钳子。注意：被解剖远离胚胎将重新生成，并应保存，以确保女性保持水润群体的组织。 </li><li>使用巴斯德吸管，将胚胎注入培养皿，安排它们平行的壁琼脂糖低谷。 </li></ol> 3，显微注射质粒DNA的<ol><li>安装在磁性底座，坐在铁板的微量。装入注射保持器，其是保持在针上的控制杆显微操作的微量的一部分。安装操纵杆操纵到铁板上，带有磁性的立场。放置在右侧解剖显微镜的这整个设置和注射夹持位置，以使得它是在观察视野中可见。 </li><li>将注入培养皿用解剖显微镜下的胚胎，定向成使得琼脂糖槽的垂直壁是到左边。将针头插入注射座，降低针尖入菜的水润媒体。 </li><li>慢慢转动顺时针方向注射器的旋钮，直到矿物油填充针和注射液源源不断的顶部观察退出针头插入液联中等。如果流过强，转动旋钮，逆时针降低压力。实行此步骤，以定期获得合适的压力，这取决于针开口的尺寸（ 即，注射针是如何被修剪的步骤1.5）。注意：如果流太强，胚胎不会注入生存。 </li><li>同时，通过解剖显微镜观察，将注入培养皿，使得第一胚胎是在视场的中心。移动针，以便它正在触摸的胚胎。用显微刺穿用针胚胎。注意：在琼脂糖槽的垂直壁将保持胚胎从一旁通过​​针推。 </li><li>使注射液流入胚胎1或2秒，然后迅速取出针。如果胚胎中有多个小区，分别注入各小区。使用镊子重新定向胚胎对准下一个单元与针的尖端。 </li><li>第一安莉芳后o为喷射，移动盘，使下胚胎是在注射位置，并重复该过程;继续下去，直到所有的胚胎被注入。 </li></ol> 4，培养和胚胎孵化<ol><li>将所有注入胚胎与几个水润有睾丸一盘（这是为了确保所有的胚胎受精）。孵化的胚胎在18℃培养箱中培养，而他们继续胚胎。 </li><li>一旦角质层阶段为止，将各注入胚胎到一个24孔板中填充有水润介质的单个井。 </li><li>孵育注入胚2周在黑暗中在18℃。 </li><li>在2周潜伏期后，检查解剖显微镜下每个注射胚胎。注意，在其中角质层期胚胎已经从小块父母组织的分离的和亲本组织已再生的任何情况。立即删除该再生性息肉，使其不英里staken一个新的幼体。 </li><li>移动注射胚胎的菜下水族灯在室温。每天检查板和收集幼体。新的幼体将白色的，因为水润息肉的典型的粉红色的颜色来自于他们所吃的丰年 。 </li><li>观察荧光显微镜下的幼体来检测转基因表达。 </li></ol>

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The authors have nothing to disclose

水润常规无性繁殖，但需要环境刺激，开始产生配子。这些刺激是不明确定义为最水润物种可能与应变的应变。 à显著障碍产生转基因水润的是获得定期的胚胎，因为它可能很难在实验室环境诱导水润成为性。 AEP的菌株25，但是，产生配子容易在实验室中，这是已经被使用至今用于制备转基因品系的唯一菌株。用于诱导配子生产中最常用的方法是通过饮食操纵?...

水润已用于研究再生，格局的形成，以及干细胞大约有250岁2 水润有由三个细胞系一个简单的身体构造：外胚层上皮，内胚层上皮，间质。所述管状体由外胚层和内胚层上皮细胞系，其中每一个是单电池层而形成的。所有的上皮细胞在体内的列是有丝分裂。当上皮细胞被移动到末端3，头部（嘴和触须）在的反口端的口端部或支脚（基底光盘），它们阻止在细胞周期的G2期和改变细胞命运4。间质细胞系的细胞位于上皮细胞之间的间隙内。此系由多能干细胞存在于身体塔5的外胚层上皮细胞层的支持。间质干细胞产生3躯体大公升型（神经，腺体细胞，nematocytes）和生殖细胞6,7。 由于刺丝胞动物门中的一员，该姐妹团所有两侧对称， 水润可以在多细胞动物中共享的基本生物过程揭示。直到最近，这些努力受到缺乏可靠的方法对基因功能的扰动阻碍。然而，随着转基因方法1的发展，我们现在能够采取水润充分利用，以更好地了解常见的多细胞动物，例如干细胞的功能，再生和构图的基本机制。转基因水润线通过注射的质粒DNA导入胚胎，这导致随机整合和表达嵌合在刚孵化的主要频率的确定。在一个特定的血统与统一表达式A线可通过无性繁殖来确定。以无性繁殖transg的能力ENIC 水润线是在大多数动物模型，这只能通过有性生殖传播的优点。此外，转基因细胞可以容易地在活体内由于动物的透明度和缺乏内源性荧光蛋白8的跟踪。 在由于第一转基因水润线七年作了1，如线已被用于各种各样的应用。在不同类型的细胞的荧光蛋白的表达使得有可能跟踪细胞运动，观察变化，细胞形态，并且追踪细胞命运都在野生型的条件和化学扰动1,5,9-12之后。此外，不同的荧光蛋白在各种细胞系表达允许特定细胞群的FAC​​S分离。这种技术已被用于干细胞特异性的mRNA和谱系特异性小RNA 13,14测序。而启动子两水润肌动蛋白基因之一得到了最广泛的应用，一些细胞类型特异性启动子已经确定，并用于驱动绿色荧光蛋白的表达在转基因水润 9,11,15,16。在未来，细胞类型特异性启动子将允许对任何特定的细胞类型的观测和收集。此外，转基因的方法已成功地用于定义WNT3子17的顺式作用调节元件。 转基因方法水润的发展提供了强有力的方法来通过异位表达，表达和击倒测试基因的功能。转基因动物已表达荧光标记的蛋白，以检查这两个功能和细胞定位18-20。此外，RNA发夹的绿色荧光蛋白转基因的3&#39;UTR的表达导致靶基因21,22击倒。在这些方法中的GFP是必需的，以确定并创立了转基因株系的追踪期间的转基因组织。但是，很可能在一些情况下，绿色荧光蛋白分子会干扰标记的蛋白质的功能。最近的研究表明， 水螅基因可以被布置在一个操纵子结构， 即，多顺反子转录物制成，然后由反式剪接前导另外分离和分开23翻译。通过将编码蛋白质或操纵子和荧光蛋白基因的下游位置的上游位置的RNA发夹的基因，可以追踪转基因组织，而无需标签编码的蛋白质或RNA发夹的基因。此方法已被用于表达与的DsRed2的操纵子结构的RNA发夹结构，以实现基因敲低14。

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">AEP Hydra Strain</td>
			<td style="width:112px;">NA</td>
			<td style="width:119px;">NA</td>
			<td style="width:576px;">Email: Celina Juliano at celina.juliano@yale.edu or Hiroshi Shimizu bubuchin2006@yahoo.co.jp</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">High Speed Maxi Kit</td>
			<td style="width:112px;">Qiagen</td>
			<td align="right" style="width:119px;">12662</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">100 x 15 mm Petri Dishes</td>
			<td style="width:112px;">BD Falcon</td>
			<td align="right" style="width:119px;">351029</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">75 x 50 mm Glass Microscope Slide</td>
			<td style="width:112px;">Sigma</td>
			<td>CLS294775X50</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Microinjection Fish Mold</td>
			<td style="width:112px;">IBI Scientific</td>
			<td style="width:119px;">FM-600</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Borosilicate Glass with Filament</td>
			<td style="width:112px;">Sutter Instrument</td>
			<td style="width:119px;">BF100-50-10</td>
			<td>O.D.: 1.0 mm, I.D.: 0.50 mm, 10 cm length</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Flaming/Brown Micropipette Puller</td>
			<td style="width:112px;">Sutter Instrument</td>
			<td style="width:119px;">P-97</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Phenol Red</td>
			<td style="width:112px;">Sigma</td>
			<td style="width:119px;">P3532</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Jewelers Forceps, Durmont #5</td>
			<td style="width:112px;">Sigma</td>
			<td>F6521</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Scalpel Blade #15</td>
			<td style="width:112px;">Fisher</td>
			<td style="width:119px;">50-822-421</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Mineral oil</td>
			<td style="width:112px;">Sigma</td>
			<td style="width:119px;">M8410-500ML</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Microinjector</td>
			<td style="width:112px;">Narishige</td>
			<td style="width:119px;">IM-9B</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Magnetic Stand</td>
			<td style="width:112px;">Narishige</td>
			<td style="width:119px;">GJ-1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Iron Plate</td>
			<td style="width:112px;">Narishige</td>
			<td style="width:119px;">IP</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Joystick Micromanipulator</td>
			<td style="width:112px;">Narishige</td>
			<td style="width:119px;">MN-151</td>
			<td></td>
		</tr>
	</tbody>
</table>

generation of transgenic hydra by embryo microinjection

As a member of the phylum Cnidaria, the sister group to all bilaterians, Hydra can shed light on fundamental biological processes shared among multicellular animals. Hydra is used as a model for the study of regeneration, pattern formation, and stem cells. However, research efforts have been hampered by lack of a reliable method for gene perturbations to study molecular function. The development of transgenic methods has revitalized the study of Hydra biology1. Transgenic Hydra allow for the tracking of live cells, sorting to yield pure cell populations for biochemical analysis, manipulation of gene function by knockdown and over-expression, and analysis of promoter function. Plasmid DNA injected into early stage embryos randomly integrates into the genome early in development. This results in hatchlings that express transgenes in patches of tissue in one or more of the three lineages (ectodermal epithelial, endodermal epithelial, or interstitial). The success rate of obtaining a hatchling with transgenic tissue is between 10% and 20%. Asexual propagation of the transgenic hatchling is used to establish a uniformly transgenic line in a particular lineage. Generating transgenic Hydra is surprisingly simple and robust, and here we describe a protocol that can be easily implemented at low cost.

建立转基因水润线饲料的转基因幼体，每2-3天用卤虫无节幼体 。稚龟孵出有时后不要吃了两天。一些新的幼体永远不会吃的，因此将无法生存。如果幼体是转基因无论是在外胚层（ 图1A）或内胚层上皮组织，让动物芽和收集有最转基因组织（ 图1B，C）的花蕾。继续与新的芽为此，直至转基因系建立与转基因中任一的外胚层（ 图1D）<...

Watch this Scientific Journal Video about Generation of Transgenic Hydra by Embryo Microinjection at JoVE.com

Generation of Transgenic Hydra by Embryo Microinjection

Stably transgenic Hydra are made by microinjection of plasmid DNA into embryos followed by random genomic integration and asexual propagation to establish a uniform line. Transgenic Hydra are used to track cell movements, overexpress genes, study promoter function, or knock down gene expression using RNAi.

转基因的产生<em&gt;水润</em&gt;通过胚胎显微注射

As a member of the phylum Cnidaria, the sister group to all bilaterians, Hydra can shed light on fundamental biological processes shared among ...

generation-of-transgenic-hydra-by-embryo-microinjection

Research

JoVE Journal

Biology

Generation of Transgenic Hydra by Embryo Microinjection

13.9K 次观看.  Yale University School of Medicine. Stably transgenic Hydra are made by microinjection of plasmid DNA into embryos followed by random genomic integration and asexual propagation to establish a uniform line. Transgenic Hydra are used to track cell movements, overexpress genes, study promoter function, or knock down gene expression using RNAi. 

视频: Generation of Transgenic Hydra by Embryo Microinjection

Microinjection of A. aegypti Embryos to Obtain Transgenic Mosquitoes

In this video, Nijole Jasinskiene demonstrates the methodology employed to generate transgenic Aedes aegypti mosquitoes, which are vectors for dengue fever.  The techniques for correctly preparing microinjection needles, dessicating embryos, and performing microinjection are demonstrated.

In this video, Nijole Jasinskiene demonstrates the methodology employed to generate transgenic Aedes aegypti mosquitoes, which are vectors for dengue fever. The techniques for correctly preparing microinjection needles, dessicating embryos, and performing microinjection are demonstrated.

埃及斑蚊胚胎显微注射获得转基因蚊子

In this video, Nijole Jasinskiene demonstrates the methodology employed to generate transgenic Aedes aegypti mosquitoes, which are vectors for dengue ...

科研

生物学

Generation of Stable Transgenic C. elegans Using Microinjection

Transgenic Caenorhabditis elegans can be readily created via microinjection of a DNA plasmid solution into the gonad 1. The plasmid DNA rearranges to form extrachromosomal concatamers that are stably inherited, though not with the same efficiency as actual chromosomes 2. A gene of interest is co-injected with an obvious phenotypic marker, such as rol-6 or GFP, to allow selection of transgenic animals under a dissecting microscope. The exogenous gene may be expressed from its native promoter for cellular localization studies. Alternatively, the transgene can be driven by a different tissue-specific promoter to assess the role of the gene product in that particular cell or tissue. This technique efficiently drives gene expression in all tissues of C. elegans except for the germline or early embryo 3. Creation of transgenic animals is widely utilized for a range of experimental paradigms. This video demonstrates the microinjection procedure to generate transgenic worms. Furthermore, selection and maintenance of stable transgenic C. elegans lines is described.

This video demonstrates the technique of microinjection into the gonad of C. elegans to create transgenic animals.

产生稳定的转基因C.使用微量注射线虫

Transgenic Caenorhabditis elegans can be readily created via microinjection of a DNA plasmid solution into the gonad 1.  The plasmid DNA rearranges to ...

Microinjection Techniques for Studying Mitosis in the Drosophila melanogaster Syncytial Embryo

This protocol describes the use of the Drosophila melanogaster syncytial embryo for studying mitosis1. Drosophila has useful genetics with a sequenced genome, and it can be easily maintained and manipulated. Many mitotic mutants exist, and transgenic flies expressing functional fluorescently (e.g. GFP) - tagged mitotic proteins have been and are being generated. Targeted gene expression is possible using the GAL4/UAS system2. 

The Drosophila early embryo carries out multiple mitoses very rapidly (cell cycle duration, &asymp;10 min). It is well suited for imaging mitosis, because during cycles 10-13, nuclei divide rapidly and synchronously without intervening cytokinesis at the surface of the embryo in a single monolayer just underneath the cortex. These rapidly dividing nuclei probably use the same mitotic machinery as other cells, but they are optimized for speed; the checkpoint is generally believed to not be stringent, allowing the study of mitotic proteins whose absence would cause cell cycle arrest in cells with a strong checkpoint. Embryos expressing GFP labeled proteins or microinjected with fluorescently labeled proteins can be easily imaged to follow live dynamics (Fig. 1). In addition, embryos can be microinjected with function-blocking antibodies or inhibitors of specific proteins to study the effect of the loss or perturbation of their function3. These reagents can diffuse throughout the embryo, reaching many spindles to produce a gradient of concentration of inhibitor, which in turn results in a gradient of defects comparable to an allelic series of mutants. Ideally, if the target protein is fluorescently labeled, the gradient of inhibition can be directly visualized4. It is assumed that the strongest phenotype is comparable to the null phenotype, although it is hard to formally exclude the possibility that the antibodies may have dominant effects in rare instances, so rigorous controls and cautious interpretation must be applied. Further away from the injection site, protein function is only partially lost allowing other functions of the target protein to become evident.

Microinjection Techniques for Studying Mitosis in the Drosophila melanogaster Syncytial Embryo

This protocol describes the use of microinjection and high resolution imaging in the Drosophila melanogaster syncytial embryo to study mitosis.

研究有丝分裂的显微注射技术果蝇合胞体胚胎

This protocol describes the use of the Drosophila melanogaster syncytial embryo  for studying mitosis1.  Drosophila has useful genetics with a sequenced ...

Production of Transgenic Xenopus laevis by Restriction Enzyme Mediated Integration and Nuclear Transplantation

Stable integration of cloned gene products into the Xenopus genome is necessary to control the time and place of expression, to express genes at later stages of embryonic development, and to define how enhancers and promoters regulate gene expression within the embryo. The protocol demonstrated here can be used to efficiently produce transgenic Xenopus laevis embryos. This transgenesis approach involves three parts: 1. Sperm nuclei are isolated from adult X. laevis testis by treatment with lysolecithin, which permeabilizes the sperm plasma membrane. 2. Egg extract is prepared by low speed centrifugation, addition of calcium to cause the extract to progress to interphase of the cell cycle, and a high-speed centrifugation to isolate interphase cytosol. 3. Nuclear transplantation: the nuclei and extract are combined with the linearized plasmid DNA to be introduced as the transgene and a small amount of restriction enzyme. During a short reaction, egg extract partially decondenses the sperm chromatin and the restriction enzyme generates chromosomal breaks that promote recombination of the transgene into the genome. The treated sperm nuclei are then transplanted into unfertilized eggs. Integration of the transgene usually occurs prior to the first embryonic cleavage such that the resulting embryos are not chimeric. These embryos can be analyzed without any need to breed to the next generation, allowing for efficient and rapid generation of transgenic embryos for analyses of promoter and gene function. Adult X. laevis resulting from this procedure also propagate the transgene through the germline and can be used to generate lines of transgenic animals for multiple purposes.

Production of Transgenic Xenopus laevis by Restriction Enzyme Mediated Integration and Nuclear Transplantation

This video protocol demonstrates a method for generating transgenic Xenopus laevis by introduction of transgenes into sperm nuclei followed by nuclear transplantation into unfertilized eggs.

生产转基因非洲爪蟾限制性内切酶介导的整合和核移植

Stable integration of cloned gene products into the Xenopus genome is necessary to control the time and place of expression, to express genes at later ...

Generation of Transgenic C. elegans by Biolistic Transformation

The number of laboratories using the free living nematode C. elegans is rapidly growing. The popularity of this biological model is attributed to a rapid generation time and short life span, easy and inexpensive maintenance, fully sequenced genome, and array of RNAi resources and mutant animals. Additionally, analysis of the C. elegans genome revealed a great similarity between worms and higher vertebrates, which suggests that research in worms could be an important adjunct to studies performed in whole mice or cultured cells. A powerful and important part of worm research is the ability to use transgenic animals to study gene localization and function. Transgenic animals can be created either via microinjection of the worm germline or through the use of biolistic bombardment. Bombardment is a newer technique and is less familiar to a number of labs. Here we describe a simple protocol to generate transgenic worms by biolistic bombardment with gold particles using the Bio-Rad PDS-1000 system. Compared with DNA microinjection into hermaphrodite germline, this protocol has the advantage of not requiring special skills from the operator with regards to identifying worm anatomy or performing microinjection. Further multiple transgenic lines are usually obtained from a single bombardment. Also in contrast to microinjection, biolistic bombardment produces transgenic animals with both extrachromosomal arrays and integrated transgenes. The ability to obtain integrated transgenic lines can avoid the use of mutagenic protocols to integrate foreign DNA. In conclusion, biolistic bombardment can be an attractive method for the generation of transgenic animals, especially for investigators not interested in investing the time and effort needed to become skilled at microinjection.

Generation of Transgenic C. elegans by Biolistic Transformation

Transgenic worms are commonly used in C. elegans research. Described is a simple, yet effective, protocol to introduce transgenes into worms using biolistic bombardment with DNA-coated gold particles. The effort involved and results of bombardment compare favorably with microinjection for the generation of transgenic animals.

生成的基因 C。线虫通过基因枪转化

The number of laboratories using the free living nematode C. elegans is rapidly growing. The popularity of this biological model is attributed to a rapid ...

Analysis of mRNA Nuclear Export Kinetics in Mammalian Cells by Microinjection

In eukaryotes, messenger RNA (mRNA) is transcribed in the nucleus and must be exported into the cytoplasm to access the translation machinery. Although the nuclear export of mRNA has been studied extensively in Xenopus oocytes1 and genetically tractable organisms such as yeast2 and the Drosophila derived S2 cell line3, few studies had been conducted in mammalian cells. Furthermore the kinetics of mRNA export in mammalian somatic cells could only be inferred indirectly4,5. In order to measure the nuclear export kinetics of mRNA in mammalian tissue culture cells, we have developed an assay that employs the power of microinjection coupled with fluorescent in situ hybridization (FISH). These assays have been used to demonstrate that in mammalian cells, the majority of mRNAs are exported in a splicing dependent manner6,7, or in manner that requires specific RNA sequences such as the signal sequence coding region (SSCR) 6. In this assay, cells are microinjected with either in vitro synthesized mRNA or plasmid DNA containing the gene of interest. The microinjected cells are incubated for various time points then fixed and the sub-cellular localization of RNA is assessed using FISH. In contrast to transfection, where transcription occurs several hours after the addition of nucleic acids, microinjection of DNA or mRNA allows for rapid expression and allows for the generation of precise kinetic data.

Here we describe an assay that employs the power of microinjection coupled with fluorescent in situ hybridization in order to accurately measure the nuclear export kinetics of mRNA in mammalian somatic cells.

在哺乳动物细胞通过显微注射mRNA的核出口动力学分析

In eukaryotes, messenger RNA (mRNA) is transcribed in the nucleus and must be exported into the cytoplasm to access the translation machinery. Although ...

Analyzing the Function of Small GTPases by Microinjection of Plasmids into Polarized Epithelial Cells

Epithelial cells polarize their plasma membrane into biochemically and functionally distinct apical and basolateral domains where the apical domain faces the 'free' surfaces and the basolateral membrane is in contact with the substrate and neighboring cells. Both membrane domains are separated by tight junctions, which form a diffusion barrier. Apical-basolateral polarization can be recapitulated successfully in culture when epithelial cells such as Madin-Darby Canine Kidney (MDCK) cells are seeded at high density on polycarbonate filters and cultured for several days 1 2. Establishment and maintenance of cell polarity is regulated by an array of small GTPases of the Ras superfamily such as RalA, Cdc42, Rab8, Rab10 and Rab13 3 4 5 6 7. Like all GTPases these proteins cycle between an inactive GDP-bound state and an active GTP-bound state. Specific mutations in the nucleotide binding regions interfere with this cycling 8. For example, Rab13T22N is permanently locked in the GDP-form and thus dubbed 'dominant negative', whereas Rab13Q67L can no longer hydrolyze GTP and is thus locked in a 'dominant active' state 7. To analyze their function in cells both dominant negative and dominant active alleles of GTPases are typically expressed at high levels to interfere with the function of the endogenous proteins 9. An elegant way to achieve high levels of overexpression in a short amount of time is to introduce the plasmids encoding the relevant proteins directly into the nuclei of polarized cells grown on filter supports using microinjection technique. This is often combined with the co-injection of reporter plasmids that encode plasma membrane receptors that are specifically sorted to the apical or basolateral domain. A cargo frequently used to analyze cargo sorting to the basolateral domain is a temperature sensitive allele of the vesicular stomatitis virus glycoprotein (VSVGts045) 10. This protein cannot fold properly at 39&deg;C and will thus be retained in the endoplasmic reticulum (ER) while the regulatory protein of interest is assembled in the cytosol. A shift to 31&deg;C will then allow VSVGts045 to fold properly, leave the ER and travel to the plasma membrane 11. This chase is typically performed in the presence of cycloheximide to prevent further protein synthesis leading to cleaner results. Here we describe in detail the procedure of microinjecting plasmids into polarized cells and subsequent incubations including temperature shifts that allow a comprehensive analysis of regulatory proteins involved in basolateral sorting.

This article details the procedures involved in overexpression and analysis of small GTPases in polarized epithelial cells using microinjection technique.

为极化上皮细胞注射质粒的小GTP酶的功能分析

Epithelial cells polarize their plasma membrane into biochemically and functionally distinct apical and basolateral domains where the apical domain faces ...

Activation of Apoptosis by Cytoplasmic Microinjection of Cytochrome c

Apoptosis, or programmed cell death, is a conserved and highly regulated pathway by which cells die1. Apoptosis can be triggered when cells encounter a wide range of cytotoxic stresses. These insults initiate signaling cascades that ultimately cause the release of cytochrome c from the mitochondrial intermembrane space to the cytoplasm2. The release of cytochrome c from mitochondria is a key event that triggers the rapid activation of caspases, the key cellular proteases which ultimately execute cell death3-4.
The pathway of apoptosis is regulated at points upstream and downstream of cytochrome c release from mitochondria5. In order to study the post-mitochondrial regulation of caspase activation, many investigators have turned to direct cytoplasmic microinjection of holocytochrome c (heme-attached) protein into cells6-9. Cytochrome c is normally localized to the mitochondria where attachment of a heme group is necessary to enable it to activate apoptosis10-11. Therefore, to directly activate caspases, it is necessary to inject the holocytochrome c protein instead of its cDNA, because while the expression of cytochrome c from cDNA constructs will result in mitochondrial targeting and heme attachment, it will be sequestered from cytosolic caspases. Thus, the direct cytosolic microinjection of purified heme-attached cytochrome c protein is a useful tool to mimic mitochondrial cytochrome c release and apoptosis without the use of toxic insults which cause cellular and mitochondrial damage.
In this article, we describe a method for the microinjection of cytochrome c protein into cells, using mouse embryonic fibroblasts (MEFs) and primary sympathetic neurons as examples. While this protocol focuses on the injection of cytochrome c for investigations of apoptosis, the techniques shown here can also be easily adapted for microinjection of other proteins of interest.

Activation of Apoptosis by Cytoplasmic Microinjection of Cytochrome c

In this protocol, we describe the direct cytoplasmic microinjection of cytochrome c protein into fibroblasts and primary sympathetic neurons. This technique allows for the introduction of cytochrome c protein into the cytoplasm of cells and mimics the release of cytochrome c from mitochondria, which occurs during apoptosis.

激活细胞凋亡的细胞色素的细胞质显微注射 C</em

Apoptosis, or programmed cell death, is a conserved and highly regulated pathway by which cells die1.  Apoptosis can be triggered when cells encounter a ...

Generation of Myospheres From hESCs by Epigenetic Reprogramming

Generation of a homogeneous and abundant population of skeletal muscle cells from human embryonic stem cells (hESCs) is a requirement for cell-based therapies and for a "disease in a dish" model of human neuromuscular diseases. Major hurdles, such as low abundance and heterogeneity of the population of interest, as well as a lack of protocols for the formation of three-dimensional contractile structures, have limited the applications of stem cells for neuromuscular disorders. We have designed a protocol that overcomes these limits by ectopic introduction of defined factors in hESCs - the muscle determination factor MyoD and SWI/SNF chromatin remodeling complex component BAF60C - that are able to reprogram hESCs into skeletal muscle cells. Here we describe the protocol established to generate hESC-derived myoblasts and promote their clustering into tridimensional miniaturized structures (myospheres) that functionally mimic miniaturized skeletal muscles7.

Here, we describe a protocol based on epigenetic reprogramming of human embryonic stem cells (hESCs) toward generating a homogeneous population of skeletal muscle progenitors that under permissive culture conditions form three-dimensional clusters of contractile myofibers (myospheres), which recapitulate biological features of human skeletal muscles.

Myospheres的产生从人类胚胎干细胞通过表观遗传修饰

Generation of a homogeneous and abundant population of skeletal muscle cells from human embryonic stem cells (hESCs) is a requirement for cell-based ...

Culturing Caenorhabditis elegans in Axenic Liquid Media and Creation of Transgenic Worms by Microparticle Bombardment

In this protocol, we present the required materials, and the procedure for making modified&#160;C. elegans&#160;Habituation and Reproduction media (mCeHR). Additionally, the steps for exposing and acclimatizing C. elegans grown on E. coli to axenic liquid media are described. Finally, downstream experiments that utilize axenic C. elegans illustrate the benefits of this procedure. The ability to analyze and determine C. elegans&#160;nutrient requirement was illustrated by growing N2 wild type worms in axenic liquid media with varying heme concentrations. This procedure can be replicated with other nutrients to determine the optimal concentration for worm growth and development or, to determine the toxicological effects of drug treatments. The effects of varied heme concentrations on the growth of wild type worms were determined through qualitative microscopic observation and by quantitating the number of worms that grew in each heme concentration. In addition, the effect of varied nutrient concentrations can be assayed by utilizing worms that express fluorescent sensors that respond to changes in the nutrient of interest. Furthermore, a large number of worms were easily produced for the generation of transgenic C. elegans using microparticle bombardment.

Culturing Caenorhabditis elegans in Axenic Liquid Media and Creation of Transgenic Worms by Microparticle Bombardment

C. elegans is usually grown on solid agar plates or in liquid cultures seeded with E. coli. To prevent bacterial byproducts from confounding toxicological and nutritional studies, we utilized an axenic liquid medium, CeHR, to grow and synchronize a large number of worms for a range of downstream applications.