我们要感谢考德威尔实验室所有成员的合作精神。运动障碍在实验室的研究已经得到了巴赫曼 - 斯特劳斯肌张力障碍及帕金森基金会，美国帕金森基金会，美国帕金森病协会，阿拉巴马州帕金森氏病协会，迈克尔J.福克斯帕金森病研究基金会，和本科生科研。

表达质粒的构建需要两个质粒：感兴趣的基因组织特异表达的第二个可选择的改造标记之一。 实验质粒<ol><li>选择一个感兴趣的组织/细胞类型，例如，神经或肌肉特异性启动子表达的启动子。如果是同一组织内的多个基因的表达，在网关系统（Invitrogen）的质粒发起人卡带都可以使用。这种方法允许任何利益recombinational克隆4子下游的基因插入，一般情况下，上游序列2-5 KB足够的正确和准确的表达。 </li><li>感兴趣的基因的cDNA克隆的方法有两种： <ol><li>传统的克隆用限制性内切酶。 </li><li>为Gateway克隆，它是使用包含网关ATT乙 recombinational序列的引物PCR扩增。扩增的cDNA第一重组到捐助载体pDONR201创建入门载体，然后转化到E.大肠杆菌菌株DH5a。入门载体是继成功的重组体的选择和小型预习DNA提取，重组到含有启动子目标的载体，以创建表达质粒。 recombinational克隆的详情，可从Invitrogen公司的网关手册或在考德威尔等5 。 </li></ol></li></ol>选择标记质粒转基因标记质粒的例子包括：ROL - 6或使用的体壁肌肉 UNC - 54启动子驱动荧光蛋白的表达。这些标记质粒通常可以从研究界内，根据请求。 微量注射线虫制备<ol><li>被注入的两个质粒DNA进行隔离。定量，并在50 ng /μL的每一个1:1的比例混合在一起。 </li><li>琼脂垫做准备： <ol><li>直到溶解在水中的2％琼脂糖溶液;加热或微波。 </li><li>在工作​​台上设置了22 × 50 mm的盖玻片。 </li><li>使用巴斯德吸管，放置在一个玻璃盖一个熔化的琼脂糖下降，立即放置在一个90度的第二个玻璃盖，在下拉到第一。重复其他几个盖玻片。夷为平地的琼脂糖光盘的直径应为15-20毫米之间。 </li><li>后琼脂糖凝固（这个只需要时刻），取出盖玻片，并允许垫完全风干（几个小时到过夜）。 </li><li>在室温下存放在玻璃盖盒垫。 </li></ol></li><li> DNA溶液的针装载机： 在中间明火加热，并迅速拉至约两倍的长度从100μL的毛细管针装载机。 毛细管冷却后的两半分开被抢购。库存10-20注射针头装载机。商店直立的离 ​​心管架。谨慎使用不刺穿自己点。 </li><li>进行显微注射​​针： 针是由一个特殊的毛细管，其中包含一个内部的玻璃纤维长丝作为DNA溶液的灯芯。这些都是拉一针拉拔机上。我们用一个Narishige PP - 830拉拔，热定型的24.8。几个针拉的时间和存储在一个小塑料盒。多针，可随时“装”上的橡皮泥地带长期储存。 </li><li>针尖“断路器”： 的显微注射针的尖端拉那么细，它实际上是在它的封闭式，并应使用玻璃盖的小碎片，破开。这些都是准备纸巾包裹在一个18 × 18毫米的玻璃盖，轻轻施加压力，直到它断裂成几件。一个有用的大小的碎片大约有3 x 4毫米。 </li><li>野生型蠕虫（N2），成长到成年早期阶段使用的标准程序6注射。准备约100正确上演蠕虫/构造注入。 </li></ol>程序<ol><li>打开主阀是连接到一个微量注射针臂和支架的氦罐。关闭调节阀，让气体进入线，使35磅的压力。注意使用脚踏板，可以释放的气体。 </li><li>一针器插入到一个标准口pipetter管（玻璃毛细管的容器中），并制定了少量的质粒混合物（1μL）。 </li><li>针器小心放入后端的注射针头，轻轻插入方式通过的长度针，直到阻力感觉。轻轻吹驱逐的DNA溶液，然后取出装载机。 </li><li>显微注射的手臂插入针头，小心地放置在小的内部橡胶垫片。 </li><li>放在琼脂板上的一个边缘的玻璃盖针破陶片。盖的碎片，以及琼脂板的整个表面，与卤烃油。将倒置显微镜舞台上的琼脂垫。 </li><li>查看使用4X的目标和明视场照明领域的中心针的位置，但还没有降低进油。此外，中心盖在玻璃碎片的内缘的位置，然后专注于IT（仍在使用4X目标）。下入油针，将玻璃盖的碎片一样焦平面。提高放大倍率切换到40X目标。重新碎片的边缘，如果有必要重新定位针尖。 </li><li>要开拓尖端的注射针头，轻轻地轻推针对盖玻璃碎片的边缘，而在同一时间，采用短脉冲的气体通过脚踏板。中针会充分破开液体的小水滴时逃脱针年底的。多余的液体流动，由于过大开幕，是不可取的，因为它会杀死动物。 </li><li>从舞台删除琼脂板，提高了针，但不改变X或Y轴位置。滑动阶段，从下面的针，取出琼脂垫，并将其放置到在解剖显微镜。垫转移到一种蠕虫病毒，油面以下。如果蠕虫蠕动，轻轻抚摩它，直到它接触琼脂垫。潮湿的蠕虫，要坚持干琼脂糖。 </li><li>注塑<ol><li>快速放置搬上舞台的琼脂垫背面，中心视野的蠕虫病毒。 </li><li>下针到蠕虫的同一平面上的重点。 </li><li>霍夫曼照明（对比度过滤器的类型）过滤器切换。这使得蠕虫的形态细节成为可见。如果Nomarski / DIC光学上倒置的范围，将工作以及。使用40X的目标，着眼于“颗粒感”合胞中心的蠕虫病毒性腺生殖核下方的“蜂窝”模式，选择定位于一侧面临的针虫的性腺是。 </li><li>微调针的位置，直到针尖还重点（性腺的“颗粒感”同一平面）。 </li><li>轻轻插入针和性腺中心申请的气体压力驱逐到性腺手臂液滴的DNA或两个简短的脉冲。你应该观察整个远端性腺的液体传播的波。不要以为更多的液体是更好，因为过多的液体流入近端性腺手臂弯曲，可以关闭卵母细胞生产。如果可能的话，根据蠕虫的位置上，以同样的方式注入其他性腺手臂。 </li><li>重要的是迅速开展工作，同时注入了一种蠕虫病毒，它可以很容易变干。注入第二性腺臂的决定后的速度有多快，你可以​​重新定位针和蠕虫依赖。整个过程应该最好少于1-2分钟。 </li></ol></li><li>从舞台上拆下的玻璃盖，放在解剖范围。应用上直接注入蠕虫（22MM KH 2 PO 4，42mm的NA 2 HPO 4，85MM氯化钠，1MM 硫酸镁 ）M9的缓冲液加入1μl（这可能需要淹没的卤烃油的表面下面的枪头）。缓冲区应补充水分的蠕虫病毒，然后将浮琼脂糖垫表面。蠕虫传输到正规板使用了蠕虫挑。琼脂糖垫可重复使用数次，直到它已成为缓冲区和蠕虫不再坚持过于饱和。 </li></ol>转基因选择<ol><li>注入蠕虫中，P 0代，被转移到个人新鲜，细菌接种板（60毫米），并允许重现。通常情况下，注入动物孵育2-3天，直到后代出现在20 ° C。 </li><li>的F 1后代观察转基因标记用荧光体视显微镜（如荧光）的证据。每个荧光，载波F 1，动物分别克隆到一个新的板块（因为每个F 1的后代，被认为是一个独特的行）。允许的F 1雌雄同体重现。 ，同样，这通常是在20 ° C为2-3天，直到后代出现。 </li><li>观察以及转基因动物的F 2代。 F 2，继承和表达的转基因阵列的动物被认为是一个“稳定” ​​行。 注：在F 1日年龄有可能是很多的转基因动物，但他们并不稳定。 F 1的转基因动物的大多数人不会传播到F 2稳定线。 注意：要控制在基因的拷贝数变异，产生三个独立的每个构造注入稳定线的最低 。 </li><li>每个独立的稳定线应保持作为一个单独的蠕虫。每一行可以传播几个转基因动物在每一代的新盘，这必须使用荧光体视显微镜，如果选择标记荧光。 </li></ol>

<ol>
	<li>Stinchcomb, D. T., Shaw, J. E., Carr, S. H., Hirsh, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extrachromosomal+DNA+transformation+of+Caenorhabditis+elegans.">Extrachromosomal DNA transformation of Caenorhabditis elegans.</a> Mol. Cell. Biol. 5, 3484-3496 (1985).</li><li>Mello, C. C., Kramer, J. M., Stinchcomb, D., Ambros, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+gene+transfer+in+C.elegans:+extrachromosomal+maintenance+and+integration+of+transforming+sequences.">Efficient gene transfer in C.elegans: extrachromosomal maintenance and integration of transforming sequences.</a> EMBO J. 10, 3959-3970 (1991).</li><li>Kelly, W. G., Xu, S., Montgomery, M. K., Fire, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distinct+requirements+for+somatic+and+germline+expression+of+a+generally+expressed+Caenorhabditis+elegans+gene.">Distinct requirements for somatic and germline expression of a generally expressed Caenorhabditis elegans gene.</a> Genetics. 146, 227-238 (1997).</li><li>Cao, S., Gelwix, C. C., Caldwell, K. A., Caldwell, G. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Torsin-mediated+neuroprotection+from+cellular+stresses+to+dopaminergic+neurons+of+C.+elegans.">Torsin-mediated neuroprotection from cellular stresses to dopaminergic neurons of C. elegans.</a> J Neurosci. 25, 3801-3812 (2005).</li><li>Caldwell, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Integrated Genomics: A Discovery-Based Laboratory Course. , (2006).</li><li>Brenner, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+genetics+of+Caenorhabditis+elegans.">The genetics of Caenorhabditis elegans.</a> Genetics. 77, 71-94 (1974).</li></ol>

执行此过程时，重要的是要记住： - 直接注入到性腺中心 - 没有注入过多的液体 - 迅速开展工作，以防止dessication。 如果进针，如它被打破过大的问题，这是最好的改造，而不是试图以比不太理想的针注入新鲜针，。 代转基因蠕虫有许多应用，如： - 突变基因的鉴定，在方法调用转化救援 - 截短蛋白的表达蛋白结构域的映射，通过 - 一个细胞和疾病的进程的基因的作用表?...

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generation of stable transgenic c. elegans using microinjection

转基因线虫可以很容易地创建，通过显微注射DNA质粒的解决方案进入性腺1。质粒DNA的重新排列，形成稳定遗传，但不以同样的效率为 2实际的染色体染色体外concatamers。一个共同感兴趣的基因注入一个明显的表型标记，如ROL - 6或GFP，在解剖显微镜下，允许转基因动物的选择。可能是外源基因表达的细胞定位研究的原始推动者。此外，转基因可以由不同的组织特异性的启动子，以评估在特定的细胞或组织的基因产物的作用。这项技术有效地推动所有组织线虫的生殖细胞或早期胚胎基因表达 3 。转基因动物的创建是广泛使用的实验范式。本视频演示了显微注射过程产生转基因的蠕虫。此外，选择和维护稳定的转基因线虫线描述。

Watch this Scientific Journal Video about Generation of Stable Transgenic C. elegans Using Microinjection at JoVE.com

Generation of Stable Transgenic C. elegans Using Microinjection

本视频演示，到线虫的性腺创建转基因动物的显微注射技术。

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 ...

generation-of-stable-transgenic-c-elegans-using-microinjection

Research

JoVE Journal

Biology

52.7K 次观看.  University of Alabama. 本视频演示，到线虫的性腺创建转基因动物的显微注射技术。

视频: Generation of Stable Transgenic C. elegans Using 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 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 ...

Imaging C. elegans Embryos using an Epifluorescent Microscope and Open Source Software

Cellular processes, such as chromosome assembly, segregation and cytokinesis,are inherently dynamic. Time-lapse imaging of living cells, using fluorescent-labeled reporter proteins or differential interference contrast (DIC) microscopy, allows for the examination of the temporal progression of these dynamic events which is otherwise inferred from analysis of fixed samples1,2. Moreover, the study of the developmental regulations of cellular processes necessitates conducting time-lapse experiments on an intact organism during development. The Caenorhabiditis elegans embryo is light-transparent and has a rapid, invariant developmental program with a known cell lineage3, thus providing an ideal experiment model for studying questions in cell biology4,5and development6-9. C. elegans is amendable to genetic manipulation by forward genetics (based on random mutagenesis10,11) and reverse genetics to target specific genes (based on RNAi-mediated interference and targeted mutagenesis12-15). In addition, transgenic animals can be readily created to express fluorescently tagged proteins or reporters16,17. These traits combine to make it easy to identify the genetic pathways regulating fundamental cellular and developmental processes in vivo18-21. In this protocol we present methods for live imaging of C. elegans embryos using DIC optics or GFP fluorescence on a compound epifluorescent microscope. We demonstrate the ease with which readily available microscopes, typically used for fixed sample imaging, can also be applied for time-lapse analysis using open-source software to automate the imaging process.

Imaging C. elegans Embryos using an Epifluorescent Microscope and Open Source Software

The C. elegans embryo is a powerful system for studying cell biology and development. We present a protocol for live imaging of C. elegans embryos utilizing DIC optics or fluorescence using readily available epifluorescent microscopes and open-source software.

成像 C。线虫胚胎使用啶显微镜和开放源码软件

Cellular processes, such as chromosome assembly, segregation and cytokinesis,are inherently dynamic. Time-lapse imaging of living cells, using ...

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 ...

Determining Genetic Expression Profiles in C. elegans Using Microarray and Real-time PCR

Synapses are composed of a presynaptic active zone in the signaling cell and a postsynaptic terminal in the target cell. In the case of chemical synapses, messages are carried by neurotransmitters released from presynaptic terminals and received by receptors on postsynaptic cells. Our previous research in Caenorhabditis elegans has shown that VSM-1 negatively regulates exocytosis. Additionally, analysis of synapses in vsm-1 mutants showed that animals lacking a fully functional VSM-1 have increased synaptic connectivity. Based on these preliminary findings, we hypothesized that C. elegans VSM-1 may play a crucial role in synaptogenesis. To test this hypothesis, double-labeled microarray analysis was performed, and gene expression profiles were determined. First, total RNA was isolated, reversely transcribed to cDNA, and hybridized to the DNA microarrays. Then, in-silico analysis of fluorescent probe hybridization revealed significant induction of many genes coding for members of the major sperm protein family (MSP) in mutants with enhanced synaptogenesis. MSPs are the major component of sperm in C. elegans and appear to signal nematode oocyte maturation and ovulation . In fruit flies, Chai and colleagues 1 demonstrated that MSP-like molecules regulate presynaptic bouton number and size at the neuromuscular junction. Moreover, analysis performed by Tsuda and coworkers 2 suggested that MSPs may act as ligands for Eph receptors and trigger receptor tyrosine kinase signaling cascades. Lastly, real time PCR analysis corroborated that the gene coding for MSP-32 is induced in vsm-1(ok1468) mutants. Taken together, research performed by our laboratory has shown that vsm-1 mutants have a significant increase in synaptic density, which could be mediated by MSP-32 signaling.

Determining Genetic Expression Profiles in C. elegans Using Microarray and Real-time PCR

Microarray analysis was conducted to determine genetic expression profiles in C. elegans, and real-time PCR was used to validate and quantify microarray data.

确定基因表达概况 C。线虫使用微阵列和实时PCR

Synapses are composed of a presynaptic active zone in the signaling cell and a postsynaptic terminal in the target cell. In the case of chemical synapses, ...

C. elegans Chemotaxis Assay

Many organisms use chemotaxis to seek out food sources, avoid noxious substances, and find mates. Caenorhabditis elegans has impressive chemotaxis behavior.
 The premise behind testing the response of the worms to an odorant is to place them in an area and observe the movement evoked in response to an odorant. Even with the many available assays, optimizing worm starting location relative to both the control and test areas, while minimizing the interaction of worms with each other, while maintaining a significant sample size remains a work in progress 1-10. The method described here aims to address these issues by modifying the assay developed by Bargmann et al.1. A Petri dish is divided into four quadrants, two opposite quadrants marked "Test" and two are designated "Control". Anesthetic is placed in all test and control sites. The worms are placed in the center of the plate with a circle marked around the origin to ensure that non-motile worms will be ignored. Utilizing a four-quadrant system rather than one 2 or two 1 eliminates bias in the movement of the worms, as they are equidistant from test and control samples, regardless of which side of the origin they began. This circumvents the problem of worms being forced to travel through a cluster of other worms to respond to an odorant, which can delay worms or force them to take a more circuitous route, yielding an incorrect interpretation of their intended path. This method also shows practical advantages by having a larger sample size and allowing the researcher to run the assay unattended and score the worms once the allotted time has expired.

C. elegans Chemotaxis Assay

A method of quantitatively evaluating the chemotactic response of Caenorhabditis elegans is described. A chemotactic index (CI) was employed as a way to precisely evaluate the response of worms to certain targets, and serve as a platform of comparison between strains and compounds of interest.

线虫趋含量的

Many organisms use chemotaxis to seek out food sources, avoid noxious substances, and find mates.  Caenorhabditis elegans has impressive chemotaxis ...

Antibody Staining in C. Elegans Using &quot;Freeze-Cracking&quot;

To stain C. elegans with antibodies, the relatively impermeable cuticle must be bypassed by chemical or mechanical methods. "Freeze-cracking" is one method used to physically pull the cuticle from nematodes by compressing nematodes between two adherent slides, freezing them, and pulling the slides apart. Freeze-cracking provides a simple and rapid way to gain access to the tissues without chemical treatment and can be used with a variety of fixatives. However, it leads to the loss of many of the specimens and the required compression mechanically distorts the sample. Practice is required to maximize recovery of samples with good morphology. Freeze-cracking can be optimized for specific fixation conditions, recovery of samples, or low non-specific staining, but not for all parameters at once. For antibodies that require very hard fixation conditions and tolerate the chemical treatments needed to chemically permeabilize the cuticle, treatment of intact nematodes in solution may be preferred. If the antibody requires a lighter fix or if the optimum fixation conditions are unknown, freeze-cracking provides a very useful way to rapidly assay the antibody and can yield specific subcellular and cellular localization information for the antigen of interest.

Antibody Staining in C. Elegans Using &quot;Freeze-Cracking&quot;

"Freeze-cracking," a method for exposing the inner tissues of the nematode C. elegans to antibodies for protein localization, is demonstrated.

抗体在染色<em&gt; C。线虫</em&gt;使用“冻结开裂”

To stain C.  elegans with antibodies, the relatively impermeable cuticle must be  bypassed by chemical or mechanical methods.  "Freeze-cracking" is one ...

Measuring the Effects of Bacteria on C. Elegans Behavior Using an Egg Retention Assay

C. elegans egg-laying behavior is affected by environmental cues such as osmolarity1 and vibration2. In the total absence of food C. elegans also cease egg-laying and retain fertilized eggs in their uterus3. However, the effect of different sources of food, especially pathogenic bacteria and particularly Enterococcus faecalis, on egg-laying
	behavior is not well characterized. The egg-in-worm (EIW) assay is a useful tool to quantify the effects of different types of bacteria, in this case E. faecalis, on egg- laying behavior.
	
	EIW assays involve counting the number of eggs retained in the uterus of C. elegans4. The EIW assay involves bleaching staged, gravid adult C. elegans to remove the cuticle and separate the retained eggs from the animal. Prior to bleaching, worms are exposed to bacteria (or any type of environmental cue) for a fixed period of time. After bleaching, one is very easily able to count the number of eggs retained inside the uterus of the worms. In this assay, a quantifiable increase in egg retention after E. faecalis exposure can be easily measured. The EIW assay is a behavioral assay that may be used to screen for potentially pathogenic bacteria or the presence of environmental toxins. In addition, the EIW assay may be a tool to screen for drugs that affect neurotransmitter signaling since egg-laying behavior is modulated by neurotransmitters such as serotonin and acetylcholine5-9.

Measuring the Effects of Bacteria on C. Elegans Behavior Using an Egg Retention Assay

An egg-in-worm (EIW) assay is a useful method to quantify egg-laying behavior. Alterations in egg laying can be a behavioral response of the model organism Caenorhabditis elegans to potentially harmful environmental substances such as those produced by pathogenic bacteria.

测量细菌的影响<em&gt; C。线虫</em&gt;行为使用一个鸡蛋保留含量

C. elegans egg-laying behavior is  affected by environmental cues such as osmolarity1 and  vibration2. In the total absence of food C. elegans also cease ...

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.

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 ...

Investigating the Spreading and Toxicity of Prion-like Proteins Using the Metazoan Model Organism C. elegans

Prions are unconventional self-propagating proteinaceous particles, devoid of any coding nucleic acid. These proteinaceous seeds serve as templates for the conversion and replication of their benign cellular isoform. Accumulating evidence suggests that many protein aggregates can act as self-propagating templates and corrupt the folding of cognate proteins. Although aggregates can be functional under certain circumstances, this process often leads to the disruption of the cellular protein homeostasis (proteostasis), eventually leading to devastating diseases such as Alzheimer&#8217;s disease (AD), Parkinson&#8217;s disease (PD), Amyotrophic lateral sclerosis (ALS), or transmissible spongiform encephalopathies (TSEs). The exact mechanisms of prion propagation and cell-to-cell spreading of protein aggregates are still subjects of intense investigation. To further this knowledge, recently a new metazoan model in Caenorhabditis elegans, for expression of the prion domain of the cytosolic yeast prion protein Sup35 has been established. This prion model offers several advantages, as it allows direct monitoring of the fluorescently tagged prion domain in living animals and ease of genetic approaches. Described here are methods to study prion-like behavior of protein aggregates and to identify modifiers of prion-induced toxicity using C. elegans.

Investigating the Spreading and Toxicity of Prion-like Proteins Using the Metazoan Model Organism C. elegans

Prion-like propagation of protein aggregates has recently emerged as being implicated in many neurodegenerative diseases. The goal of this protocol is to describe, how to use the nematode C. elegans as a model system to monitor protein spreading and to investigate prion-like phenomena.

调查的传播和毒性朊病毒样蛋白使用后生动物模式生物<em&gt;温度。线虫</em

Prions are unconventional self-propagating proteinaceous particles, devoid of any coding nucleic acid. These proteinaceous seeds serve as templates for ...