Nous tenons à souligner l&#39;esprit de coopération de tous les membres du laboratoire Caldwell. Mouvements anormaux de recherche dans le laboratoire a été soutenu par la dystonie Bachmann-Strauss &amp; Parkinson Foundation, Royaume-Parkinson Foundation, American Association Maladie de Parkinson, la maladie de Parkinson Association de l&#39;Alabama, la Fondation Michael J. Fox pour la recherche sur le Parkinson, et une recherche de premier cycle

A. Construction plasmide d&#39;expression Deux plasmides sont nécessaires: un pour l&#39;expression tissu-spécifique du gène d&#39;intérêt et un second en tant que marqueur sélectionnable de transformation (bien que le marqueur de plasmide est habituellement disponible au sein de la communauté de la recherche). Experimental plasmidique <ol><li> Sélectionnez un promoteur qui est exprimé dans le type de tissu / cellule d&#39;intérêt, dans ce cas, le transporteur DA (EPEC-1) promoteur est utilisé. Ce plasmide d&#39;expression a été créé comme une passerelle Invitrogen système compatible vecteur de destination (pDEST-DAT-1) pour permettre l&#39;insertion d&#39;un gène d&#39;intérêt en aval du promoteur par clonage par recombinaison 1. </li><li> L&#39;ADNc du gène d&#39;intérêt est amplifié par PCR utilisant des amorces qui contiennent la passerelle att séquence B recombinaison. L&#39;ADNc amplifié est d&#39;abord recombinés dans le vecteur pDONR201 des donateurs pour créer le vecteur d&#39;entrée et ensuite transformé en E. coli souche DH5a. Après la sélection de recombinants succès et mini-prep isolation de l&#39;ADN, le vecteur d&#39;entrée est recombinée dans le pDEST-DAT-1 vecteurs de destination pour créer le plasmide d&#39;expression. Détails sur le clonage par recombinaison sont disponibles à partir du manuel soit Passerelle Invitrogen ou dans Caldwell et al. 5. </li></ol> Marqueurs de sélection plasmidique Un marqueur transgénique plasmide est constitué d&#39;un vecteur par un promoteur qui contrôlent l&#39;expression d&#39;une protéine fluorescente dans un tissu clair. Dans cette procédure particulière, le promoteur unc-54 entraîne l&#39;expression des protéines dans le corps de cerises paroi musculaire (ponctua-54:: cerise). B. Génération de C. elegans transgéniques via micro-injection Voir l&#39;article relatif JoVE: <a href="https://www.jove.com/index/details.stp?ID=833">https://www.jove.com/index/details.stp?ID=833</a> C. Analyse génétique des croix pour la neurodégénérescence DA <ol><li> Worms sont cultivés en utilisant des procédures standard de 6. </li><li> Placer 10 EPEC-1:: α-syn; EPEC-1:: GFP mâles 1, 2 sur une plaque d&#39;accouplement avec 3-4 L4 hermaphrodites transgéniques étape (exprimant EPEC-1: gène X et de ponctua-54:: cerise) . Cela devrait être effectué individuellement pour chacune des trois lignes séparées stables créés. Après deux jours, retirez les mâles. </li><li> Inspectez la génération F1; s&#39;il ya plusieurs descendants mâles, l&#39;accouplement a réussi. </li><li> Clone sur 5 hermaphrodites L4 animaux de chaque croix que présentent à la fois l&#39;EPEC-1:: GFP marqueur fluorescent (hérité du parent mâle) et la ponctua-54:: cerise marqueur fluorescent muscles du corps mur (hérité du parent hermaphrodite). Les animaux clonés doivent être au stade L4 afin de s&#39;assurer qu&#39;ils n&#39;ont pas encore accouplées avec des mâles présents sur le plateau. Permettez-leur de l&#39;auto-cross et produire une descendance F2. </li><li> Animaux F2 produits à l&#39;étape 3 sont clonées sur. Plus précisément, le transfert des animaux ~ 50-10 qui présentent deux marqueurs fluorescents pour leurs propres assiettes individuelles. Écran de la génération F3 pour les plaques d&#39;où 100% de la GFP animaux expriment en DA neurones et certains des animaux sont d&#39;exprimer cerise dans les cellules du corps muscle de la paroi. Ces animaux seront homozygotes pour l&#39;EPEC-1:: α-syn; EPEC-1: gène de la GFP alors stablement transmission du transgène nouvellement créé (gène X). </li></ol> D. Analyse dopaminergiques Neuron <ol><li> Préparer une plaque de frais pour chacune des trois lignes créées ci-dessus, ainsi que l&#39;EPEC-1:: α-syn; EPEC-1:: GFP seule souche. Leur permettre de croître à l&#39;âge adulte. </li><li> Transfert 30-40 adultes transgénique à partir de chaque ligne sur une plaque fraîche. Permettez-leur de jeter des embryons pendant 4 heures à 20 º C. </li><li> Retirez les adultes. Autoriser les embryons à développer pendant 3-4 jours à 20 º C jusqu&#39;à ce qu&#39;ils atteignent le stade L4. </li><li> Choisissez ~ 100 transgénique L4 animaux à une plaque contenant 0,04 mg / ml de 5-fluoro-2&#39;-désoxyuridine (FUDR). Ce nucléotide de développement analogiques des blocs de la prochaine génération par inhibition de la synthèse de l&#39;ADN, empêchant ainsi la descendance de l&#39;animal de laboratoire écrasante 7. Protéger les plaques FUDR de la lumière car il est sensible à la lumière. </li><li> Les animaux adultes seront analysés à plusieurs jours suivant la ponte. Les jours d&#39;analyse appropriées sont déterminées empiriquement. Par exemple, nous avons déterminé que 77%, 87% et 90% de l&#39;EPEC-1:: α-syn; EPEC-1:: GFP animaux présentent dégénéré neurones DA aux jours 6, 7 et 10 de développement post, respectivement. Par conséquent, si nous prévoyons que le transgène d&#39;intérêt pourrait renforcer la neurodégénérescence, nous allons analyser au jour 6 et / ou 7. De même, des transgènes qui pourrait supprimer la neurodégénérescence sera analysée au jour 7 et 10. </li><li> Faire 4 nouveaux pads agarose (contrairement à des plaquettes de gélose microinjection, elles sont utilisées fraîches et ne sont pas autorisés à sécher). Énoncer deux lames de microscope qui ont un morceau de ruban à travers eux; le ruban sert entretoise pour les plaquettes équivalente d&#39;épaisseur. Lay tiersglisser entre eux sur le banc (ils sont positionnés sur trois au courant). Déposer une goutte de agarose fondu sur la lame du centre et rapidement mettre une autre lame sur le dessus de l&#39;agarose, perpendiculaire à et à travers tous les trois diapositives. Faites plusieurs pads supplémentaires, laissant les deux diapositives ensemble jusqu&#39;à ce que le pad est prêt à l&#39;emploi. </li><li> Pour analyser le ver neurones DA, déposer une goutte 6 pi de 3 mM lévamisole (un anesthésique) sur un 22 x 30 mm couvercle en verre. Choisissez 40 animaux adultes (10 supplémentaires au-delà des 30 à être marqué) dans la goutte, puis inverser le couvercle en verre sur le tampon d&#39;agarose. Répétez l&#39;opération pour chacune des autres lignes. </li><li> Score 30 animaux par ligne pour la présence de chaque CEP et ADE neurone à l&#39;aide d&#39;un microscope composé avec épifluorescence et un cube de filtre qui permet la visualisation de FITC ou GFP. Nous utilisons un filtre à gagner à la GFP HyQ cube (technologie Chroma). Enregistrer les données sur la feuille de pointage ci-joint. Animaux de type sauvage conservera fluorescence de la GFP complète dans tous les CEP 4 et 2 neurones ADE. Les animaux individuels présentant une perte de neurones DA sont marqués comme non-WT ou &quot;dégénérer&quot;. De même, la mesure de la dégénérescence peut être marqué par le comptage des neurones perdus au total dans chaque animal ainsi que la population. </li><li> Répétez l&#39;analyse 2 fois plus (30 animaux plus par ligne et par essai). Le nombre total de vers de type sauvage présentant des neurones DA à partir des trois tours des analyses est de moyenne. L&#39;analyse statistique pour la neuroprotection est ensuite effectuée à l&#39;aide de l&#39;étudiant t-test (p &lt;0,05) pour comparer les vers de contrôle (EPEC-1:: α-syn; EPEC-1:: GFP) par des souches qui les gènes candidats sur-expriment dans les neurones DA . </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>

La perte liée à l&#39;âge des neurones à dopamine est une caractéristique clinique de la maladie de Parkinson et a été associée à l&#39;accumulation ou mauvais repliement d&#39;une protéine appelée alpha-synucléine. Ici, nous montrer comment l&#39;étiquette, par l&#39;intermédiaire d&#39;un transgène fluorescentes, les neurones dopaminergiques de C. elegans et imiter la neurodégénérescence observée dans la maladie de Parkinson en coexprimant humaine alpha-synucléine dans ces cellules. Cette vidéo illust...

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<td>Agarose</td>
<td>Ultrapure</td>
<td>Invitrogen</td>
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application of a c. elegans dopamine neuron degeneration assay for the validation of potential parkinson's disease genes

Les améliorations apportées au diagnostic et au traitement de la maladie de Parkinson (MP) sont tributaires des connaissances sur les facteurs qui rendent la susceptibilité des populations à risque. Dans le processus de tenter d&#39;identifier de nouveaux facteurs génétiques associés à la DP, les scientifiques ont généré de nombreuses listes de gènes candidats, les polymorphismes, et les protéines qui représentent des avancées importantes, mais ces pistes restent mécanistiquement indéfini. Notre travail vise vers un rétrécissement de manière significative ces listes en exploitant les avantages d&#39;un système de modèle animal simple. Bien que les humains ont des milliards de neurones, le nématode Caenorhabditis elegans microscopiques a précisément 302, dont huit seulement de produire la dopamine (DA) dans hemaphrodites. Expression d&#39;un gène humain codant pour la protéine PD-associé, l&#39;alpha-synucléine, dans C. elegans résultats neurones DA de la dose et âge-dépendante neurodégénérescence. Worms exprimer humaine alpha-synucléine dans les neurones DA sont isogéniques et d&#39;exprimer à la fois la GFP et l&#39;alpha-synucléine humaine sous le promoteur du transporteur DA (EPEC-1). La présence de la GFP sert de marqueur pour suivre facilement visualisés neurodégénérescence DA chez ces animaux. Nous avons d&#39;abord démontré que l&#39;alpha-synucléine neurodégénérescence induite DA ont pu être sauvés dans ces animaux par torsinA, une protéine à activité chaperon moléculaire 1. En outre, le candidat PD-liés gènes identifiés dans notre laboratoire au travers de grands efforts de dépistage ARNi en utilisant un dosage repliement alpha-synucléine ont ensuite été sur-exprimée dans les neurones DA C. elegans. Nous avons déterminé que cinq des sept gènes testés représenté modulateurs candidat significatif de PD comme ils sauvé l&#39;alpha-synucléine induite DA neurodégénérescence 2. Par ailleurs, le laboratoire de Lindquist (cette question de Jupiter) a effectué la levure écrans où l&#39;alpha-synucléine-dépendant toxicité est utilisée comme une lecture des gènes qui peuvent augmenter ou supprimer la cytotoxicité. Nous avons ensuite examiné les gènes candidats de la levure dans notre test de C. elegans neurodégénérescence alpha-synucléine-induite et validé avec succès plusieurs de ces trois cibles, 4. Notre méthodologie implique la génération d&#39;un vecteur d&#39;expression de C. elegans DA neurone-spécifique en utilisant le clonage par recombinaison des gènes candidats ADNc sous le contrôle du promoteur EPEC-1. Ces plasmides sont ensuite microinjectés de type sauvage (N2) vers, avec un marqueur de sélection pour la transformation réussie. Plusieurs lignées transgéniques stables produisant la protéine candidate dans les neurones DA sont obtenus indépendamment et ensuite franchi dans la souche alpha-synucléine dégénératives et évalués pour la neurodégénérescence, à la fois l&#39;animal et le niveau des neurones individuels, au cours du vieillissement.

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&#039;s Disease Genes

Cette vidéo montre comment utiliser C. elegans afin d&#39;évaluer la neurodégénérescence neurone dopaminergique comme un modèle pour la maladie de Parkinson. Par ailleurs, cribles génétiques sont utilisés pour identifier les facteurs qui accentuent la dégénérescence ou sont neuroprotecteurs.

Application d&#39;une Assay C. elegans dégénérescence des neurones dopaminergiques pour la validation du potentiel de la maladie de Parkinson gènes

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

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

15.9K vues.  University of Alabama. Cette vidéo montre comment utiliser C. elegans afin d'évaluer la neurodégénérescence neurone dopaminergique comme un modèle pour la maladie de Parkinson. Par ailleurs, cribles génétiques sont utilisés pour identifier les facteurs qui accentuent la dégénérescence ou sont neuroprotecteurs.

Vidéo: 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&#039;s Disease

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

Ole Isacson: développement de nouvelles thérapies pour la maladie de Parkinson

Recherche

Biologie

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.

Génération de Stable elegans transgéniques C. Utilisation de microinjection

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.

Murine Model for Parkinson&#039;s Disease: from 6-OH Dopamine Lesion to Behavioral Test

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.

Modèle murin de la maladie de Parkinson: de lésion dopamine 6-OH au test comportemental

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.

Génération de souris transgéniques C. elegans Par transformation biolistique

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.

La production de C. elegans Les transgènes via Recombineering avec le GalK Marqueurs de sélection

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.

Chromosomics: détection d&#39;altérations numériques et structurelles dans les 24 chromosomes de l&#39;homme en utilisant simultanément un dosage FISH Novel OctoChrome

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.

En utilisant l&#39;ARN médiée par la stratégie d&#39;alimentation interférences à l&#39;écran pour des gènes impliqués dans le règlement de la taille du corps du nématode<em&gt; C. elegans</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.

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.

Un procédé de culture embryonnaire<em&gt; C. elegans</em&gt; Cellules

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.

Mesurer les effets des bactéries sur<em&gt; C. Elegans</em&gt; Comportement aide d&#39;un test de rétention d&#39;oeufs

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 ...