Wir wollen den Geist der Zusammenarbeit aller Caldwell Lab Mitglieder anzuerkennen. Bewegungsstörungen Forschung im Labor wurde von der Bachmann-Strauss Dystonie &amp; Parkinson Foundation, Vereinigte Parkinson Foundation, American Parkinson Disease Association, Parkinson Disease Association of Alabama, die Michael J. Fox Foundation for Parkinson Research, und ein Undergraduate Research unterstützt

A. Expressionsplasmid Construction Zwei Plasmide sind erforderlich: eine für Gewebe-spezifische Expression des Gens von Interesse und eine zweite als wählbare Transformation Marker (obwohl die Marker-Plasmid ist in der Regel innerhalb der Forschergemeinschaft zur Verfügung). Experimentelle Plasmid <ol><li> Wählen Sie einen Promotor, der in dem Gewebe / Zelltyp von Interesse zum Ausdruck kommt, in diesem Fall, der DA-Transporter (PDAT-1) Promotor verwendet wird. Das Expressionsplasmid wurde als Invitrogen Gateway-System-kompatiblen Destination-Vektor (pDEST-DAT-1) geschaffen, um die Einfügung eines Gens von Interesse stromabwärts des Promotors durch recombinational Klonen 1 zu ermöglichen. </li><li> Die cDNA des Gens von Interesse ist die PCR mit den Primern, dass das Gateway att B recombinational Sequenz enthalten. Die amplifizierte cDNA wird zunächst in der Donor-Vektor pDONR201 um den Eintrag zu erstellen Vektor rekombiniert und dann in E. coli-Stamm DH5. Nach der Auswahl von erfolgreichen Rekombinanten und mini-prep Isolierung von DNA, wird der Eintrag Vektor in die pDEST-DAT-1 Zielvektor zu schaffen Expressionsplasmid rekombiniert. Details zu recombinational Klonen sind entweder von der Invitrogen Gateway-Handbuch oder in Caldwell et al Verfügung. 5. </li></ol> Selektionsmarker Plasmid Eine transgene Marker Plasmid besteht aus einem Vektor mit einem Promotor, der die Expression eines fluoreszierenden Proteins in eine offensichtliche Gewebe. In diesem speziellen Verfahren, treibt das unc-54-Promotor Kirsche Proteinexpression in Körperwand Muskel (punktiert-54:: Kirsche). B. Herstellung von transgenen C. elegans über Mikroinjektion Siehe zugehörige JoVE Artikel: <a href="https://www.jove.com/index/details.stp?ID=833">https://www.jove.com/index/details.stp?ID=833</a> C. Genetische Kreuze für DA Neurodegeneration Analysis <ol><li> Worms angebaut werden mit Standard-Verfahren 6. </li><li> Platz 10 PDAT-1:: α-syn; PDAT-1:: GFP Rüden 1, 2 auf eine Gegenplatte mit 3-4 L4 Stadium transgenen Hermaphroditen (Ausdruck PDAT-1:: Gen-X und punktiert-54:: Kirsche) . Diese sind individuell für jede der drei separaten stabile Linien erstellt durchgeführt werden. Nach zwei Tagen, entfernen Sie die Männchen. </li><li> Überprüfen Sie die F1-Generation, wenn es mehrere männliche Nachkommen sind, war die Paarung erfolgreich. </li><li> Clone out 5 Hermaphrodit L4 Tiere von jeder Kreuz, das zeigen sowohl die PDAT-1:: GFP fluoreszierenden Marker (geerbt von den männlichen Elternteil) und die punktiert-54:: cherry fluoreszierenden Körper Wand Muskel-Marker (geerbt von der Hermaphrodit Elternteil). Die geklonten Tiere müssen auf der L4 Stadium werden, um sicherzustellen, dass sie noch nicht mit männlichen Tieren auf dem Teller gedeckt. Erlauben Sie ihnen, sich selbst kreuzen und produzieren F2-Nachkommen. </li><li> F2 Tiere in Schritt 3 erzeugt werden geklont aus. Insbesondere Transfer ~ 5-10 Tieren, die sowohl fluoreszierende Marker aufweisen, ihre eigenen individuellen Platten. Bildschirm der F3 Generation für Platten, wo 100% der Tiere zum Ausdruck GFP in DA Neuronen und einige der Tiere zum Ausdruck Kirsche in Körperwand Muskelzellen. Diese Tiere werden homozygot für die PDAT-1:: α-syn; PDAT-1:: GFP-Gen, während stabil Übertragung der neu geschaffenen Transgen (Gen X). </li></ol> D. dopaminergen Neuronen Analysis <ol><li> Bereiten Sie eine neue Platte für jede der drei Linien oben erstellt, sowie die PDAT-1:: α-syn; PDAT-1:: GFP allein belasten. Lassen Sie sie bis zum Erwachsenenalter wachsen. </li><li> Transfer 30-40 transgenen Erwachsene aus jeder Zeile auf eine frische Platte. Erlauben Sie ihnen, Embryonen für 4 Stunden lag bei 20 º C. </li><li> Entfernen Sie die Erwachsenen. Lassen Sie die Embryonen für 3-4 Tage bei 20 º C zu entwickeln, bis sie die L4 Stadium zu erreichen. </li><li> Wählen Sie ~ 100 transgenen L4 Tiere eine Platte mit 0,04 mg / ml 5-Fluor-2&#39;-desoxyuridin (FUDR). Diese Nukleotidanalog Blöcke Entwicklung der nächsten Generation über eine Hemmung der DNA-Synthese und verhindert so den Nachkommen von überwältigender den experimentellen Tiere 7. Schützen Sie die FUDR Platten aus dem Licht, da sie lichtempfindlich ist. </li><li> Erwachsene Tiere werden an verschiedenen Tagen nach der Eiablage analysiert werden. Die entsprechenden Tage Analyse werden empirisch ermittelt. Zum Beispiel haben wir festgestellt, dass 77%, 87% und 90% der PDAT-1:: α-syn; PDAT-1:: GFP Tiere zeigen degenerierte DA Neuronen an den Tagen 6, 7 und 10 nach der Entwicklung bzw.. Wenn wir also davon aus, dass das Transgen von Interesse sein könnten Neurodegeneration zu verbessern, werden wir am Tag 6 und / oder 7 zu analysieren. Ebenso werden Transgene, die Neurodegeneration zu unterdrücken könnte an den Tagen 7 und 10 analysiert werden. </li><li> Machen Sie 4 frische Agarose-Pads (im Gegensatz zu den Mikroinjektion Agar-Pads, diese werden frisch und dürfen nicht austrocknen). Set Sie zwei Objektträger, dass ein Stück Klebeband haben über sie, die Band dient als Abstandshalter für äquivalent dicken Ballen. Lay ein DrittelRutsche zwischen ihnen auf der Bank (sie sind drei nebeneinander positioniert). Geben Sie einen Tropfen geschmolzenen Agarose auf die Mitte schieben und schnell lag eine andere Folie auf der Oberseite des Agarose, senkrecht und quer durch alle drei Dias. Machen Sie mehrere zusätzliche Pads, so dass die beiden Schieber zusammen, bis das Pad einsatzbereit ist. </li><li> Zur Analyse Wurm DA Neuronen, statt einer 6 ul Rückgang von 3 mM Levamisol (Narkose) auf einem 22 x 30 mm Deckglas. Wählen Sie 40 erwachsenen Tieren (10 zusätzliche jenseits der 30 zu sein hat) in den Tropfen, dann kehren die Deckglas auf das Agarose-Pad. Wiederholen Sie für jede der anderen Linien. </li><li> Ergebnis 30 Tiere pro Zeile auf das Vorhandensein von jedem CEP und ADE Neuron mit einem zusammengesetzten Mikroskop mit Epifluoreszenz und einen Filter Würfel, der Visualisierung von FITC oder GFP ermöglicht. Wir verwenden ein Endow GFP HYQ Filter Würfel (Chroma Technology). Rekord Daten auf den angeschlossenen Auswertungsbogen. Wild-Typ Tiere behalten die volle GFP-Fluoreszenz in allen 4 CEP und 2 ADE Neuronen. Einzelne Tiere ausstellen Verlust der DA Neurone sind als Nicht-WT oder &quot;entartet&quot; gewertet. Ebenso kann das Ausmaß der Degeneration durch Zählen der insgesamt Neuronen in jedem Tier sowie der Bevölkerung verloren gewertet. </li><li> Wiederholen Sie die Analyse 2 weitere Male (30 weitere Tiere pro Zeile pro Versuch). Die Gesamtzahl der Würmer ausstellenden Wildtyp-DA Neuronen aus den drei Runden der Analysen gemittelt wird. Statistische Analyse zur Neuroprotektion wird dann unter Verwendung des Student-t-Test (p &lt;0,05) zur Kontrolle Würmer (PDAT-1:: α-syn; PDAT-1:: GFP) zu vergleichen mit den Stämmen, die über-express Kandidatengene in DA Neuronen . </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>

Die altersabhängige Verlust von Dopamin-Neuronen ist ein klinisches Kennzeichen der Parkinson-Krankheit und hat mit der Akkumulation oder Fehlfaltung eines Proteins namens Alpha-Synuclein in Verbindung gebracht. Hier zeigen wir, wie Label, über eine fluoreszierende Transgen, die dopaminergen Neuronen von C. elegans und Mimik der Neurodegeneration bei Parkinson-Erkrankung, die durch Koexpression humanes Alpha-Synuclein zu sehen in diesen Zellen. Dieses Video zeigt die Methodik für Wachstum, genetische Kreuzung, Montage un...

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<td>Agarose</td>
<td>Ultrapure</td>
<td>Invitrogen</td>
<td>15510-027</td>
<td>&nbsp;</td>
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<td>Coverglass 20x30 mm</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>12-548-5A</td>
<td>&nbsp;</td>
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<td>Microscope Slides</td>
<td>Plain, 3x1"</td>
<td>Fisher</td>
<td>12-549</td>
<td>&nbsp;</td>
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<td>Dissecting with Fluorescence</td>
<td>Microscope</td>
<td>Nikon</td>
<td>SMZ800</td>
<td>&nbsp;</td>
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<td>Dissecting</td>
<td>Microscope</td>
<td>Nikon</td>
<td>SMZ645</td>
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<td>Microscope</td>
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application of a c. elegans dopamine neuron degeneration assay for the validation of potential parkinson's disease genes

Verbesserungen bei der Diagnose und Behandlung der Parkinson-Krankheit (PD) sind abhängig von Wissen über die Anfälligkeit Faktoren, die Bevölkerung zu machen in Gefahr. In den Prozess der Versuch, neue genetische Faktoren mit PD verbunden zu identifizieren, haben Wissenschaftler viele Listen von Kandidaten-Gene, Polymorphismen, und Proteine, die wichtige Fortschritte darstellen erzeugt, aber diese führt bleiben mechanistisch undefined. Unsere Arbeit richtet sich deutlich verengt solche Listen unter Ausnutzung der Vorteile einer einfachen Tiermodell System abzielen. Während Menschen Milliarden von Neuronen haben, hat die mikroskopische Fadenwurm Caenorhabditis elegans genau 302, von denen nur acht produzieren Dopamin (DA) in hemaphrodites. Expression eines menschlichen Gens für den PD-assoziierten Protein, alpha-Synuclein, in C. elegans DA Neuronen führt zu Dosierung und altersabhängige Neurodegeneration. Worms zum Ausdruck menschlichen Alpha-Synuclein in DA Neuronen isogenen und exprimieren sowohl GFP und humanes Alpha-Synuclein unter der DA-Transporter-Promotor (PDAT-1). Die Anwesenheit von GFP dient als leicht visualisiert Marker für folgende DA Neurodegeneration bei diesen Tieren. Wir haben uns zunächst gezeigt, dass Alpha-Synuclein-induzierte DA Neurodegeneration bei diesen Tieren konnte durch torsinA, ein Protein mit molekularer Chaperone Aktivität 1 gerettet werden. Weitere, Kandidat PD-assoziierter Gene in unserem Labor über eine groß angelegte RNAi-Screening Bemühungen mit einem Alpha-Synuclein Fehlfaltung Assay identifiziert wurden dann in C. elegans DA Neuronen überexprimiert. Wir haben festgestellt, dass fünf der sieben Gene vertreten signifikanten Kandidaten Modulatoren von PD getestet, wie sie alpha-Synuclein induzierten Neurodegeneration DA 2 gerettet. Darüber hinaus hat die Lindquist Lab (diese Ausgabe von JoVE) Hefe-Bildschirme durchgeführt, wobei Alpha-Synuclein-abhängige Toxizität als Anzeige für die Gene, die zu verbessern oder zu unterdrücken Zytotoxizität verwendet wird. Wir Anschließend prüfte die Hefe Kandidatengene in unserem C. elegans alpha-Synuclein induzierten Neurodegeneration Assay und erfolgreich validiert viele dieser Ziele 3, 4. Unsere Methodik umfasst Erzeugung eines C. elegans DA Neuronen-spezifische Expressionsvektor mit recombinational Klonen von Kandidatengen cDNAs unter der Kontrolle der PDAT-1-Promotor. Diese Plasmide werden dann in Wildtyp-(N2) Würmer mikroinjiziert, zusammen mit einem Selektionsmarker für eine erfolgreiche Transformation. Mehrere stabilen transgenen Linien Herstellung der Kandidaten-Protein in DA Neuronen erhalten und dann unabhängig gekreuzt in die alpha-Synuclein degenerative Belastung und bewertet für Neurodegeneration, sowohl an der Tier-und individuellen Neuron Ebene, im Laufe des Alterns.

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

Dieses Video zeigt, wie C. elegans zu dopaminergen Neuronen Neurodegeneration als Modell für die Parkinson-Krankheit zu beurteilen. Darüber hinaus sind genetische Screens verwendet, um Faktoren, die entweder zu verstärken Degeneration oder neuroprotektive identifizieren.

Die Anwendung eines C. elegans Dopamin Neuron Degeneration Assay für die Validierung von potenziellen Parkinson Genes

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

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

15.9K Aufrufe.  University of Alabama. Dieses Video zeigt, wie C. elegans zu dopaminergen Neuronen Neurodegeneration als Modell für die Parkinson-Krankheit zu beurteilen. Darüber hinaus sind genetische Screens verwendet, um Faktoren, die entweder zu verstärken Degeneration oder neuroprotektive identifizieren.

Serie: 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: Entwicklung neuer Therapien zur Behandlung der Parkinson-Krankheit

Forschung

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.

Generierung von stabilen transgenen C. elegans mit Mikroinjektion

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.

Mausmodell für Morbus Parkinson: von 6-OH Dopamin Läsion Behavioral-Test

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.

Erzeugung von transgenen C. elegans Nach 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 ...

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.

Die Produktion von C. elegans Transgene via Recombineering mit dem GalK Selektionsmarker

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: Erkennung von numerischen und strukturellen Veränderungen in alle 24 menschlichen Chromosomen gleichzeitig unter Verwendung eines neuartigen OctoChrome FISH-Test

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.

Mit RNA-Interferenz Feeding Strategy to Screen for Genes in Body Größe der Rechtsetzung in der Nematode Beteiligte<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.

C. elegans Chemotaxis-Assay

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.

Ein Verfahren zur Kultivierung von embryonalen<em&gt; C. elegans</em&gt; Zellen

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.

Messung der Auswirkungen von Bakterien auf<em&gt; C. Elegans</em&gt; Verhalten Mit einem Ei 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 ...