转基因的产生<em>水润</em>通过胚胎显微注射
Summary
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.
Abstract
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.
Introduction
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FACS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'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。
Protocol
Representative Results
Discussion
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?…
Declarações
The authors have nothing to disclose.
Acknowledgements
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。
Materials
| AEP Hydra Strain | NA | NA | Email: Celina Juliano at celina.juliano@yale.edu or Hiroshi Shimizu bubuchin2006@yahoo.co.jp |
| High Speed Maxi Kit | Qiagen | 12662 | |
| 100 x 15 mm Petri Dishes | BD Falcon | 351029 | |
| 75 x 50 mm Glass Microscope Slide | Sigma | CLS294775X50 | |
| Microinjection Fish Mold | IBI Scientific | FM-600 | |
| Borosilicate Glass with Filament | Sutter Instrument | BF100-50-10 | O.D.: 1.0 mm, I.D.: 0.50 mm, 10 cm length |
| Flaming/Brown Micropipette Puller | Sutter Instrument | P-97 | |
| Phenol Red | Sigma | P3532 | |
| Jewelers Forceps, Durmont #5 | Sigma | F6521 | |
| Scalpel Blade #15 | Fisher | 50-822-421 | |
| Mineral oil | Sigma | M8410-500ML | |
| Microinjector | Narishige | IM-9B | |
| Magnetic Stand | Narishige | GJ-1 | |
| Iron Plate | Narishige | IP | |
| Joystick Micromanipulator | Narishige | MN-151 |
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