Name: application of a c. elegans dopamine neuron degeneration assay for the validation of potential parkinson's disease genes
Uploaded: 2008-07-18T23:30:00
Description: 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

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?...

<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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<td>Agarose</td>
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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 (Video) | JoVE

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 ...

Research

JoVE Journal

Biology

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: Development of New Therapies for Parkinson's Disease (Video) | JoVE

OLE Isacson：帕金森氏病的新疗法的发展

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生物学

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

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