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

A.表达质粒的构建需要两个质粒：感兴趣的基因组织特异性表达和作为第二个可选择的改造标记（虽然通常是由研究界内的标记质粒）之一。 实验质粒<ol><li>选择一个利益的组织/细胞类型中表达的启动子，在这种情况下，DA转运（Pdat 1）发起人为使用。这表达质粒作为Invitrogen公司的网关系统兼容的目标向量（pDEST - DAT - 1），允许插入任何发起人的利益下游基因的 recombinational克隆1。 </li><li>感兴趣的基因的cDNA PCR扩增用引物包含网关ATT乙recombinational序列。扩增的cDNA第一重组到捐助载体pDONR201创建入门载体，然后转化到大肠杆菌菌株DH5α中。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pDEST - DAT - 1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>选择标记质粒转基因标记质粒组成一个驾驶了一个明显的组织中的荧光蛋白表达的启动子的载体。在这个特殊的过程中，UNC - 54启动子驱动樱桃体壁肌肉蛋白的表达（Punc - 54：樱花）。 B.通过显微注射转基因C. elegans的的代请参阅相关的朱庇特的<a href="https://www.jove.com/index/details.stp?ID=833">文章</a> ：https://www.jove.com/index/details.stp?ID=833 C.遗传横渡DA神经退行性疾病分析<ol><li>蠕虫是种植使用的标准程序。 </li><li>放置10 Pdat - 1：α- SYN; Pdat - 1：GFP的男性1，2到一个3-4 L4阶段转基因雌雄同体交配板（表达Pdat 1：基因X和Punc - 54：樱花） 。这应该是单独为每个创建三个单独的稳定线。两天后，取出男性。 </li><li> F1代的检查，如果有几个男的后代，交配成功。 </li><li>克隆出5雌雄同体L4从每个交叉动物展览Pdat - 1：GFP荧光标记（父本继承）和Punc - 54：樱花荧光体壁肌肉标记（雌雄同体父继承）。克隆动物需要在L4阶段，以确保他们尚未与板块上存在的雄性动物交配。让他们自交产生F2代。 </li><li>在第3步中产生的F2动物克隆。具体地说，转移到自己的个人板，表现出两种荧光标记5-10动物。屏幕板，其中100％的动物表达绿色荧光蛋白在DA能神经元和一些动物在体壁肌肉细胞中都表达樱桃F3代。这些动物将Pdat - 1纯合子：α- SYN; Pdat - 1：绿色荧光蛋白基因，而稳定的传输新创建的转基因（基因X）。 </li></ol> D.多巴胺能神经元分析<ol><li>准备新鲜板，以及为每个上面创建的三线Pdat - 1：α- SYN; Pdat - 1：GFP的单独应变。让他们长到成年。 </li><li>从每行传送到一个​​新的板块30-40的转基因成年人。允许他们打下了4个小时的胚胎在20℃ </li><li>取出的成年人。允许胚胎为3-4天，在20℃开发，直到他们达到的L4阶段。 </li><li>选择板含有0.04 mg / ml的5 - 氟-2&#39; - 脱氧尿苷（FUDR）〜100的转基因L4动物。这核苷酸模拟模块通过抑制DNA合成的下一代的发展，从而防止铺天盖地而来的7个实验动物的后代。光保护FUDR板，因为它是光敏感。 </li><li>成年动物进行分析，在产蛋后的各种天。凭经验确定适当的分析天。例如，我们已经确定，77％，87％和90％的Pdat - 1：α- SYN; Pdat - 1：GFP的动物表现出堕落的DA能神经元，在6天，7日和10后的发展。因此，如果我们预测，转基因可能会提高神经退行性疾病的利益，我们将分析，在第6天和/或7。同样，将可能抑制神经退行性疾病的转基因分析，在7和10天。 </li><li>使4个新鲜的琼脂糖凝胶垫（与显微注射琼脂垫不同的是，这些都是使用新鲜的，不得干出）。设置了两个显微镜幻灯片，在它们之间有一块的磁带;磁带作为等价脚垫厚实间隔。奠定了第三个它们之间在板凳上滑动​​（它们定位三排）。广场中心幻灯片上的熔化琼脂糖下降，并迅速奠定琼脂糖上另一张幻灯片，垂直和所有三张幻灯片。使一些额外的垫，留下两个幻灯片，直到垫准备使用。 </li><li>要分析蠕虫DA能神经元，地方上一个22 × 30毫米的玻璃盖的3毫米左旋咪唑（一种麻醉剂）6μL下降。挑选40个成年动物（超出30 10额外得分）下降，然后翻转到琼脂糖垫玻璃盖。其他各线的重复。 </li><li>分数每行30只动物，为每个CEP和ADE神经使用epifluorescence和过滤器的多维数据集，允许可视化FITC或GFP的复合式显微镜的存在。我们使用一个赋予GFP HYQ过滤立方体（色度科技）。附加的评分表上的记录数据。野生动物将保留在所有4 CEP的完整的绿色荧光和2 ADE的神经元。参展的DA能神经元的损失动物个体取得非WT或“堕落”。同样，变性程度上可以取得，通过计算每个动物以及人口失去了总神经元。 </li><li>重复2次以上（30多个动物每人审判）的分析。参展三轮分析野生型的DA能神经元的蠕虫总数的平均值。神经保护的统计分析是，然后执行使用的学生的t检验（P &lt;0.05），以比较控制蠕虫（Pdat - 1：：α- SYN; Pdat - 1：：GFP）与株DA能神经元的过度表达候选基因。 </li></ol>

<ol>
	<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>Hamamichi, S., Rivas, R. N., Knight, A. L., Cao, S., 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=Hypothesis-based+RNAi+screening+identifies+neuroprotective+genes+in+a+Parkinson&#x2019;s+disease+model.">Hypothesis-based RNAi screening identifies neuroprotective genes in a Parkinson&#x2019;s disease model.</a> PNAS. 105, 728-733 (2008).</li><li>Cooper, A. A., Gitler, A. D., Cashikar, A., Haynes, C. M., Hill, K. J., Bhullar, B., Liu, K., Xu, K., Strathearn, K. E., Liu, F., Cao, S., Caldwell, K. A., Caldwell, G. A., Marsischky, G., Kolodner, R. D., Labaer, J., Rochet, J. C., Bonini, N. M., Lindquist, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=alpha-synuclein+blocks+ER-golgi+traffic+and+Rab1+rescues+neuron+loss+in+Parkinson's+models.">alpha-synuclein blocks ER-golgi traffic and Rab1 rescues neuron loss in Parkinson's models.</a> Science. 313, 324-328 (2006).</li><li>Gitler, A. D., Bevis, B. J., Shorter, J., Strathearn, K. E., Hamamichi, S., Su, L. J., Caldwell, K. A., Caldwell, G. A., Rochet, J. -. C., McCaffery, J. M., Barlowe, C., Lindquist, 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+Parkinson's+disease+protein+alpha-synuclein+disrupts+cellular+Rab+homeostasis.">The Parkinson's disease protein alpha-synuclein disrupts cellular Rab homeostasis.</a> PNAS. 105, 145-150 (2008).</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=Integrated+Genomics:+A+Discovery-Based+Laboratory+Course.">Integrated Genomics: A Discovery-Based Laboratory Course.</a> , (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><li>Gandhi, S., Santelli, J., Mitchell, J. D. H., Stiles, J. W., Sanadi, 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=A+simple+method+for+maintaining+large,+aging+populations+of+Caenorhabditis+elegans.">A simple method for maintaining large, aging populations of Caenorhabditis elegans.</a> Mechanisms of Ageing and Development. 12, 137-150 (1980).</li></ol>

年龄依赖性的多巴胺神经元的损失是帕金森氏病的临床标志，并已积累或称为α-突触核蛋白的一种蛋白质的错误折叠有关。在这里，我们将演示如何标注，通过荧光的转基因，C的多巴胺能神经元elegans和模仿在这些细胞中的共表达人α-突触核蛋白在帕金森氏症的神经退行性疾病。 此影片描绘的增长，遗传隧道，安装，和转基因线虫的得分来评估遗传因素，提高神经退行性疾病，或提供在老化过...

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application of a c. elegans dopamine neuron degeneration assay for the validation of potential parkinson's disease genes

帕金森氏病（PD）的诊断和治疗的改进取决于关于在高危人群的易感因素，呈现的知识。在试图找出与PD相关的新的遗传因素的过程中，科学家们产生了许多候选基因，多态性，蛋白质，代表了重要进展列出，但这些信息仍然机械地不确定。我们的工作目的是利用一个简单的动物模型系统的优势，如列出走向显着缩小。虽然人类有十亿的神经元，微观蛔虫线虫正是302，其中只有八个产生多巴胺（DA），在hemaphrodites。表达一个人的PD相关蛋白，α-突触核蛋白基因编码线虫DA能神经元剂量与年龄相关的神经退行性疾病的结果。 蠕虫DA能神经元中表达人α-突触核蛋白等基因和表达GFP和人α-突触核蛋白的DA转运子（Pdat 1）下。绿色荧光蛋白的存在，作为一个在这些动物中的DA神经退行性疾病容易可视化的标记。我们初步表明，α-突触核蛋白诱导DA神经退行性疾病，可以在这些动物中救出torsinA，具有分子伴侣活性的蛋白质1。此外，候选人的PD相关基因的鉴定，我们通过大规模RNAi筛选使用α-突触核蛋白的错误折叠检测工作的实验室，然后在C. elegans的DA能神经元过度表达。我们决定，七五的基因测试，因为他们救出α-突触核蛋白诱导DA神经退行性疾病 2 PD的显着代表候选人调制。此外，林德基斯特实验室（这个问题的朱庇特）已经完成酵母屏幕，使依赖α-突触核蛋白毒性是用于读出一个基因，可以增强或抑制细胞毒性。其后，我们研究线虫α-突触核蛋白诱导神经退行性疾病检测酵母候选基因，并成功地验证这些目标的3， 4。 我们的方法涉及一个线虫DA神经元特异性表达Pdat - 1启动子的控制下使用的候选基因的cDNA recombinational克隆载体的一代。然后，这些质粒显微注射野生型（N2），蠕虫，伴随着一个成功转型的可选标志。多个稳定生产的DA能神经元的候选蛋白的转基因株系的获得，然后自主划线到α- synuclein的退行性应变和神经退行性疾病进行评估，在动物和单个神经元的水平，在老化的过程。

Watch this Scientific Journal Video about Application of a C. elegans Dopamine Neuron Degeneration Assay for the Validation of Potential Parkinson's Disease Genes at JoVE.com

Application of a C. elegans Dopamine Neuron Degeneration Assay for the Validation of Potential Parkinson's Disease Genes

该视频演示如何使用线虫作为帕金森病模型的多巴胺能神经元的神经退行性疾病，以评估。此外，遗传屏幕是用来识别，要么提高变性或神经因素。

一个线虫多巴胺神经元退化含量的应用验证潜在的帕金森氏症基因

Improvements to the diagnosis and treatment of Parkinson's disease (PD) are dependent upon knowledge about susceptibility factors that render populations ...

application-c-elegans-dopamine-neuron-degeneration-assay-for

Research

JoVE Journal

Biology

16.0K 次观看.  University of Alabama. 该视频演示如何使用线虫作为帕金森病模型的多巴胺能神经元的神经退行性疾病，以评估。此外，遗传屏幕是用来识别，要么提高变性或神经因素。

视频: Application of a C. elegans Dopamine Neuron Degeneration Assay for the Validation of Potential Parkinson's Disease Genes

Ole Isacson: Development of New Therapies for Parkinson's Disease

Ole Isacson gives a concise overview of Parkinsons's disease, its causes, therapeutic strategies, and advances in Parkinson's research.

OLE Isacson：帕金森氏病的新疗法的发展

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 ...

Murine Model for Parkinson's Disease: from 6-OH Dopamine Lesion to Behavioral Test

Parkinson's disease (PD) affects at least 6.5 million people worldwide, irrespective of gender, social, ethnic, economic, or geographic boundaries. Key symptoms, such as tremor, rigidity and bradikinesia, develop when about 3/4 of dopaminergic cells are lost in the substantia nigra, and fail to provide for the smooth, coordinated regulation of striatal motor circuits. Depression and hallucinations are common, and dementia eventually occurs in 20% of patients. At this time, there is no treatment to delay or stop the progression of PD. Rather, the medications currently available aim more towards the alleviation of these symptoms. New surgical strategies may reversibly switch on the functionally damaged circuits through the electrical stimulation of deep brain structures, but although deep brain stimulation is a major advance, it is not suitable for all patients. It remains therefore necessary to test new cell therapy approaches in preclinical models.
Selective neurotoxic disruption of dopaminergic pathways can be reproduced by injection of 6-hydroxydopamine (6-OHDA) or MPTP (1-methyl-4-phenyl-1,2,3,6-tertahydropyridine) whereas depleting drugs and oxidative-damaging chemicals may also reproduce specific features of PD in rodents. Unlike MPTP, 6-OHDA lesions cause massive irreversible neuronal loss, and can be uni- or bilateral. The 6-OHDA lesion model is reliable, leads to robust motor deficits, and is the most widely used after 40 years of research in rats1. As interactions between grafted cells and host can now be studied more thoroughly in mice rather than in rats, the model has been transposed to mice2,3, where it has been recently characterized4.
In this video, we demonstrate how to lesion the left nigro-striatal pathway of anesthetized mice by slowly delivering 2.0 &mu;L of 6-OHDA through a stereotaxically inserted micro-syringe needle. The loss of dopaminergic input occurs within days, and the functional impairments can be monitored over post-operative weeks and months by rating animal rotations induced by dopaminergic agents5. Here, we show full-body contralateral rotations occurring 10 minutes after a single subcutaneous administration of apomorphine, measured one month after the lesion. Outcomes and drawbacks are discussed below.

Parkinson disease is caused by loss of dopaminergic innervation to the striatum, which can be experimentally induced by 6-OH-dopamine. We describe how to perform a stereotaxic lesion and to monitor apomorphine-induced rotational behavior in mice. This model is useful and reliable for testing new therapies for Parkinson disease.

帕金森氏病的小鼠模型：从6 - OH多巴胺病变行为测试

Parkinson's disease (PD) affects at least 6.5 million people worldwide, irrespective of gender, social, ethnic, economic, or geographic boundaries. Key ...

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 ...

The Production of C. elegans Transgenes via Recombineering with the galK Selectable Marker

The creation of transgenic animals is widely utilized in C. elegans research including the use of GFP fusion proteins to study the regulation and expression pattern of genes of interest or generation of tandem affinity purification (TAP) tagged versions of specific genes to facilitate their purification. Typically transgenes are generated by placing a promoter upstream of a GFP reporter gene or cDNA of interest, and this often produces a representative expression pattern. However, critical elements of gene regulation, such as control elements in the 3' untranslated region or alternative promoters, could be missed by this approach. Further only a single splice variant can be usually studied by this means. In contrast, the use of worm genomic DNA carried by fosmid DNA clones likely includes most if not all elements involved in gene regulation in vivo which permits the greater ability to capture the genuine expression pattern and timing. To facilitate the generation of transgenes using fosmid DNA, we describe an E. coli based recombineering procedure to insert GFP, a TAP-tag, or other sequences of interest into any location in the gene. The procedure uses the galK gene as the selection marker for both the positive and negative selection steps in recombineering which results in obtaining the desired modification with high efficiency. Further, plasmids containing the galK gene flanked by homology arms to commonly used GFP and TAP fusion genes are available which reduce the cost of oligos by 50% when generating a GFP or TAP fusion protein. These plasmids use the R6K replication origin which precludes the need for extensive PCR product purification. Finally, we also demonstrate a technique to integrate the unc-119 marker on to the fosmid backbone which allows the fosmid to be directly injected or bombarded into worms to generate transgenic animals. This video demonstrates the procedures involved in generating a transgene via recombineering using this method.

The Production of C. elegans Transgenes via Recombineering with the galK Selectable Marker

The ability to produce transgenes for Caenorhabditis elegans using genomic DNA carried by fosmids is particularly attractive as all of the native regulatory elements are retained. Described is a simple and robust procedure for the production of transgenes via recombineering with the galK selectable marker.

生产 C。线虫</em通过重组工程与转基因 galK选择标记

The creation of transgenic animals is widely utilized in C. elegans research including the use of GFP fusion proteins to study the regulation and ...

Chromosomics: Detection of Numerical and Structural Alterations in All 24 Human Chromosomes Simultaneously Using a Novel OctoChrome FISH Assay

Fluorescence in situ hybridization (FISH) is a technique that allows specific DNA sequences to be detected on metaphase or interphase chromosomes in cell nuclei1. The technique uses DNA probes with unique sequences that hybridize to whole chromosomes or specific chromosomal regions, and serves as a powerful adjunct to classic cytogenetics. For instance, many earlier studies reported the frequent detection of increased chromosome aberrations in leukemia patients related with benzene exposure, benzene-poisoning patients, and healthy workers exposed to benzene, using classic cytogenetic analysis2. Using FISH, leukemia-specific chromosomal alterations have been observed to be elevated in apparently healthy workers exposed to benzene3-6, indicating the critical roles of cytogentic changes in benzene-induced leukemogenesis. 
Generally, a single FISH assay examines only one or a few whole chromosomes or specific loci per slide, so multiple hybridizations need to be conducted on multiple slides to cover all of the human chromosomes. Spectral karyotyping (SKY) allows visualization of the whole genome simultaneously, but the requirement for special software and equipment limits its application7. Here, we describe a novel FISH assay, OctoChrome-FISH, which can be applied for Chromosomics, which we define here as the simultaneous analysis of all 24 human chromosomes on one slide in human studies, such as chromosome-wide aneuploidy study (CWAS)8. The basis of the method, marketed by Cytocell as the Chromoprobe Multiprobe System, is an OctoChrome device that is divided into 8 squares, each of which carries three different whole chromosome painting probes (Figure 1). Each of the three probes is directly labeled with a different colored fluorophore, green (FITC), red (Texas Red), and blue (Coumarin). The arrangement of chromosome combinations on the OctoChrome device has been designed to facilitate the identification of the non-random structural chromosome alterations (translocations) found in the most common leukemias and lymphomas, for instance t(9;22), t(15;17), t(8;21), t(14;18)9. Moreover, numerical changes (aneuploidy) in chromosomes can be detected concurrently. The corresponding template slide is also divided into 8 squares onto which metaphase spreads are bound (Figure 2), and is positioned over the OctoChrome device. The probes and target DNA are denatured at high-temperature and hybridized in a humid chamber, and then all 24 human chromosomes can be visualized simultaneously. 
OctoChrome FISH is a promising technique for the clinical diagnosis of leukemia and lymphoma and for detection of aneuploidies in all chromosomes. We have applied this new Chromosomic approach in a CWAS study of benzene-exposed Chinese workers8,10.

A novel fluorescence in situ hybridization (FISH) method that simultaneously examines both numerical and structural chromosome alterations, particularly the specific chromosomal translocations associated with leukemia and lymphoma, of all 24 human chromosomes on a single device in one hybridization, is described.

在所有人类染色体24 chromosomics:检测的数值模拟和结构上的改动同时使用一种新型OctoChrome鱼含量

Fluorescence in situ hybridization (FISH) is a technique that allows specific DNA sequences to be detected on metaphase or interphase chromosomes in cell ...

Using RNA-mediated Interference Feeding Strategy to Screen for Genes Involved in Body Size Regulation in the Nematode C. elegans

Double-strand RNA-mediated interference (RNAi) is an effective strategy to knock down target gene expression1-3. It has been applied to many model systems including plants, invertebrates and vertebrates. There are various methods to achieve RNAi in vivo4,5. For example, the target gene may be transformed into an RNAi vector, and then either permanently or transiently transformed into cell lines or primary cells to achieve gene knockdown effects; alternatively synthesized double-strand oligonucleotides from specific target genes (RNAi oligos) may be transiently transformed into cell lines or primary cells to silence target genes; or synthesized double-strand RNA molecules may be microinjected into an organism. Since the nematode C. elegans uses bacteria as a food source, feeding the animals with bacteria expressing double-strand RNA against target genes provides a viable strategy6. Here we present an RNAi feeding method to score body size phenotype. Body size in C. elegans is regulated primarily by the TGF- &beta; - like ligand DBL-1, so this assay is appropriate for identification of TGF-&beta; signaling components7. We used different strains including two RNAi hypersensitive strains to repeat the RNAi feeding experiments. Our results showed that rrf-3 strain gave us the best expected RNAi phenotype. The method is easy to perform, reproducible, and easily quantified. Furthermore, our protocol minimizes the use of specialized equipment, so it is suitable for smaller laboratories or those at predominantly undergraduate institutions.

Using RNA-mediated Interference Feeding Strategy to Screen for Genes Involved in Body Size Regulation in the Nematode C. elegans

We demonstrate how to use the RNAi feeding technique to knock down target genes and score body size phenotype in C. elegans. This method could be used for a large scale screen to identify potential genetic components of interest, such as those involved in body size regulation by DBL-1/TGF-&beta; signaling.

使用屏幕在车身尺寸规例中的线虫基因介导的RNA干扰取食策略<em&gt; C.线虫</em

Double-strand RNA-mediated interference (RNAi) is an effective strategy to knock down target gene expression1-3. It has been applied to many model systems ...

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 ...

A Method for Culturing Embryonic C. elegans Cells

C. elegans is a powerful model system, in which genetic and molecular techniques are easily applicable. Until recently though, techniques that require direct access to cells and isolation of specific cell types, could not be applied in C. elegans. This limitation was due to the fact that tissues are confined within a pressurized cuticle which is not easily digested by treatment with enzymes and/or detergents. Based on early pioneer work by Laird Bloom, Christensen and colleagues 1 developed a robust method for culturing C. elegans embryonic cells in large scale. Eggs are isolated from gravid adults by treatment with bleach/NaOH and subsequently treated with chitinase to remove the eggshells. Embryonic cells are then dissociated by manual pipetting and plated onto substrate-covered glass in serum-enriched media. Within 24 hr of isolation cells begin to differentiate by changing morphology and by expressing cell specific markers. C. elegans cells cultured using this method survive for up 2 weeks in vitro and have been used for electrophysiological, immunochemical, and imaging analyses as well as they have been sorted and used for microarray profiling.

A Method for Culturing Embryonic C. elegans Cells

We describe here a revised protocol for large-scale culture of embryonic C. elegans cells. Embryonic C. elegans cells cultured in vitro using this method, appear to differentiate and recapitulate the expression of genes in a cell specific manner. Techniques that require direct access to the cells or isolation of specific cell types from the other tissues can be applied on C. elegans cultured cells.

一种用于培养胚胎<em&gt; C。线虫</em&gt;细胞

C. elegans is a powerful model system, in which genetic and molecular techniques are easily applicable. Until recently though, techniques that require ...

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 ...

一个线虫多巴胺神经元退化含量的应用验证潜在的帕金森氏症基因

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此视频中的章节

OLE Isacson：帕金森氏病的新疗法的发展

产生稳定的转基因C.使用微量注射线虫

帕金森氏病的小鼠模型：从6 - OH多巴胺病变行为测试

生成的基因 C。线虫通过基因枪转化

生产 C。线虫 galK选择标记

在所有人类染色体24 chromosomics:检测的数值模拟和结构上的改动同时使用一种新型OctoChrome鱼含量

使用屏幕在车身尺寸规例中的线虫基因介导的RNA干扰取食策略

线虫趋含量的

一种用于培养胚胎

测量细菌的影响