Vogliamo riconoscere lo spirito di cooperazione di tutti i membri Lab Caldwell. Disturbi del movimento di ricerca in laboratorio è stato sostenuto dalla Bachmann-Strauss Fondazione Parkinson e distonia, Regno Parkinson Foundation, American Parkinson Disease Association, il morbo di Parkinson Associazione dell&#39;Alabama, Michael J. Fox Foundation for Parkinson Research, e una ricerca di laurea

A. Costruzione Espressione plasmidi Due plasmidi sono obbligatori: uno per l&#39;espressione tessuto-specifica del gene di interesse e un secondo come marcatore trasformazione selezionabili (anche se il marcatore plasmide è solitamente disponibile all&#39;interno della comunità di ricerca). Sperimentale plasmidi <ol><li> Selezionare un promotore che si esprime nel tessuto / cellula tipo di interesse, in questo caso, il trasportatore della DA (Pdat-1) promotore viene utilizzato. Questa espressione plasmide è stato creato come un gateway Invitrogen compatibili con il sistema vettoriale destinazione (pDEST-DAT-1) per consentire l&#39;inserimento di un gene di interesse a valle del promotore di clonazione ricombinazione 1. </li><li> Il cDNA del gene di interesse è amplificato mediante PCR utilizzando primers che contengono il Gateway att sequenza B ricombinazione. Il cDNA amplificato viene prima ricombinato in un vettore donatore pDONR201 per creare il vettore di ingresso e poi trasformato in DH5α ceppo di E. coli. A seguito di selezione dei ricombinanti successo e mini-prep isolamento del DNA, il vettore di ingresso viene ricombinato in pDEST-DAT-1 vettore di destinazione per creare il plasmide di espressione. Dettagli sulla clonazione ricombinazione sono disponibili sia dal manuale Gateway Invitrogen o in Caldwell et al. 5. </li></ol> Marker selezionabile plasmidi Un indicatore transgenico plasmide è costituito da un vettore con un promotore di guida l&#39;espressione di una proteina fluorescente in un tessuto ovvio. In questa particolare procedura, l&#39;unc-54 promotore unità espressione ciliegia proteina nel muscolo del corpo parete (Punc-54:: ciliegio). B. Generazione di transgenici C. elegans tramite microiniezione Vedi relativi JOVE articolo: <a href="https://www.jove.com/index/details.stp?ID=833">https://www.jove.com/index/details.stp?ID=833</a> C. per l&#39;analisi genetica attraversa neurodegenerazione DA <ol><li> I worm sono coltivati ​​utilizzando le procedure standard 6. </li><li> Place 10 Pdat-1:: α-syn; Pdat-1:: GFP maschi 1, 2 su un piatto di accoppiamento con ermafroditi fase 3-4 L4 transgenici (che esprime Pdat-1:: X gene e Punc-54:: ciliegio) . Questo dovrebbe essere eseguito singolarmente per ciascuna delle tre linee separate stabile creato. Dopo due giorni, togliere i maschi. </li><li> Ispezionare la generazione F1, se ci sono diversi progenie di sesso maschile, l&#39;accoppiamento ha avuto successo. </li><li> Clone fuori 5 ermafrodita L4 animali da ogni croce che presentano sia il Pdat-1:: marcatore fluorescente GFP (ereditato dal genitore di sesso maschile) e la Punc-54:: ciliegio muro marcatore fluorescente corpo muscolare (ereditati dal genitore ermafrodita). Gli animali clonati bisogno di essere in fase di L4 per garantire che non hanno ancora accoppiati con animali maschi presenti sulla piastra. Consentire loro di auto-cross e produrre progenie F2. </li><li> Animali F2 prodotte in fase 3 vengono clonati fuori. In particolare, il trasferimento di animali ~ 5-10 che presentano entrambi i marcatori fluorescenti ai loro piatti individuali. Schermo la generazione F3 per piatti dove il 100% degli animali esprimere GFP in neuroni DA e alcuni degli animali sono espressi ciliegio nel corpo di cellule muscolari della parete. Questi animali saranno omozigoti per la Pdat-1:: α-syn; Pdat-1:: gene GFP mentre stabilmente la trasmissione del transgene appena creato (gene X). </li></ol> D. dopaminergici Neuron Analisi <ol><li> Preparare un piatto fresco per ciascuna delle tre linee creato in precedenza, così come la Pdat-1:: α-syn; Pdat-1:: GFP solo ceppo. Permettere loro di crescere fino all&#39;età adulta. </li><li> Trasferimento 30-40 adulti transgenici da ogni riga su un piatto fresco. Permettere loro di porre embrioni per 4 ore a 20 ° C. </li><li> Rimuovere gli adulti. Permettono di sviluppare gli embrioni per 3-4 giorni a 20 ° C fino a raggiungere la fase di L4. </li><li> Scegli ~ 100 L4 animali transgenici per un piatto contenente 0,04 mg / ml di 5-fluoro-2&#39;-deossiuridina (FUDR). Questo nucleotide sviluppo analogica blocchi della prossima generazione attraverso l&#39;inibizione della sintesi del DNA, impedendo così la discendenza dalla travolgente animali da esperimento 7. Proteggere le piastre FUDR dalla luce in quanto è sensibile alla luce. </li><li> Animali adulti saranno analizzati a diversi giorni dopo la deposizione delle uova. I giorni appropriato di analisi sono determinati empiricamente. Per esempio, abbiamo determinato che il 77%, 87% e il 90% del Pdat-1:: α-syn; Pdat-1:: GFP animali mostrano degenerato neuroni DA nei giorni 6, 7 e 10 di sviluppo post, rispettivamente. Pertanto, se si prevede che il transgene di interesse potrebbero aumentare neurodegenerazione, analizzeremo al giorno 6 e / o 7. Allo stesso modo, transgeni che potrebbero sopprimere neurodegenerazione saranno analizzati a giorni 7 e 10. </li><li> Fai 4 pastiglie fresca agarosio (a differenza di pastiglie agar la microiniezione, questi vengono utilizzati freschi e non sono autorizzati ad asciugarsi). Ha definito due vetrini da microscopio che hanno un pezzo di nastro attraverso loro, il nastro serve come distanziatore per cuscinetti equivalente di spessore. Lay terzoscivolare tra di loro in panchina (sono posizionati tre al passo). Mettere una goccia di agarosio fuso sul vetrino centro e rapidamente porre un altro scivolo in cima alla agarosio, perpendicolare e in tutti e tre diapositive. Fare alcune pastiglie aggiuntive, lasciando i due scivoli insieme fino a quando il pad è pronto all&#39;uso. </li><li> Per analizzare verme neuroni DA, posto un calo del 6 ml di 3 levamisolo mM (un anestetico) su un x 22 30 mm di vetro di copertura. Scegli 40 animali adulti (10 in più oltre i 30 ad essere segnati) nella goccia, poi invertire il vetro di copertura sul pad agarosio. Ripetere per ciascuna delle altre linee. </li><li> Punteggio 30 animali per riga per la presenza di ogni CEP e ADE neurone utilizzando un microscopio composto con epifluorescenza e un cubo filtro che permette la visualizzazione di FITC o GFP. Usiamo un Dotare GFP HYQ filtro cubo (la tecnologia Chroma). Registrare dati sul foglio di punteggio allegata. Wild-type animali mantiene fluorescenza GFP completa in tutti e 4 CEP e 2 neuroni ADE. Singoli animali presentano perdita di neuroni DA vengono segnati come non WT o di &quot;degenerazione&quot;. Allo stesso modo, il grado di degenerazione può essere segnata contando i neuroni perdita totale di ogni animale e la popolazione. </li><li> Ripetere l&#39;analisi altre 2 volte (30 animali più per riga per ogni prova). Il numero totale di vermi espositrici wild-type neuroni DA dai tre turni di analisi è media. L&#39;analisi statistica per la neuroprotezione viene eseguita utilizzando il t di Student-test (p &lt;0,05) per confrontare i vermi di controllo (Pdat-1:: α-syn; Pdat-1:: GFP) con ceppi che l&#39;eccesso di esprimere geni candidati nei neuroni 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>

L&#39;età-dipendente la perdita dei neuroni dopaminergici è una caratteristica clinica del morbo di Parkinson ed è stata associata con l&#39;accumulo o misfolding di una proteina chiamata alfa-sinucleina. Qui mostriamo come etichettare, attraverso un transgene fluorescente, i neuroni dopaminergici di C. elegans e imitare la neurodegenerazione nella malattia di Parkinson da coexpressing umano alfa-sinucleina in queste cellule. Questo video illustra la metodologia per la crescita, incrocio genetico, montaggio, e il puntegg...

<!DOCTYPE html PUBLIC "-//W3C//DTD XHTML 1.0 Transitional//EN" "http://www.w3.org/TR/xhtml1/DTD/xhtml1-transitional.dtd">
<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Agarose</td>
<td>Ultrapure</td>
<td>Invitrogen</td>
<td>15510-027</td>
<td>&nbsp;</td>
<tr>
<td>Coverglass 20x30 mm</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>12-548-5A</td>
<td>&nbsp;</td>
<tr>
<td>Microscope Slides</td>
<td>Plain, 3x1"</td>
<td>Fisher</td>
<td>12-549</td>
<td>&nbsp;</td>
<tr>
<td>Dissecting with Fluorescence</td>
<td>Microscope</td>
<td>Nikon</td>
<td>SMZ800</td>
<td>&nbsp;</td>
<tr>
<td>Dissecting</td>
<td>Microscope</td>
<td>Nikon</td>
<td>SMZ645</td>
<td>&nbsp;</td>
<tr>
<td>Epifluorescent</td>
<td>Microscope</td>
<td>Nikon</td>
<td>Model E-800</td>
<td>&nbsp;</td>
<tr>
<td>Filter Cube, GFP HYQ</td>
<td>Endow Bandpass</td>
<td>Chroma Technology</td>
<td>&nbsp;</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

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

Miglioramenti per la diagnosi e il trattamento della malattia di Parkinson (PD) dipendono le conoscenze sui fattori di suscettibilità che rendono le popolazioni a rischio. Nel processo di tentare di identificare nuovi fattori genetici associati a PD, gli scienziati hanno generato molte liste di geni candidati, polimorfismi, e le proteine ​​che rappresentano importanti passi avanti, ma questi cavi rimangono meccanicisticamente indefinito. Il nostro lavoro si rivolge in modo significativo restringimento verso tali elenchi, sfruttando i vantaggi di un sistema semplice modello animale. Mentre gli esseri umani hanno miliardi di neuroni, il microscopico verme Caenorhabditis elegans ha precisamente 302, di cui solo otto producono dopamina (DA) in hemaphrodites. Espressione di un gene umano che codifica per il PD-proteina associata, l&#39;alfa-sinucleina, in C. elegans DA risultati neuroni nel dosaggio e dipendente dall&#39;età neurodegenerazione. Worm che esprimono umano alfa-sinucleina nei neuroni DA vengono isogeniche ed esprimono sia la GFP e umano alfa-sinucleina sotto il promotore trasportatore DA (Pdat-1). La presenza di GFP serve come marcatore per seguire facilmente visualizzati neurodegenerazione DA in questi animali. Inizialmente abbiamo dimostrato che l&#39;alfa-sinucleina indotta neurodegenerazione DA potrebbe essere salvato in questi animali da torsinA, una proteina con attività di chaperone molecolare 1. Inoltre, candidato PD-correlati geni identificati nel nostro laboratorio attraverso grandi sforzi di screening RNAi con un alfa-sinucleina saggio misfolding sono stati poi sovra-espresso in C. elegans DA neuroni. Abbiamo determinato che cinque dei sette geni testati rappresentato modulatori candidato significative del PD in quanto salvato alfa-sinucleina indotta DA neurodegenerazione 2. Inoltre, il Laboratorio Lindquist (questo numero di Giove) si è esibito schermi lievito per cui alfa-sinucleina-dipendente tossicità è usato come una lettura di geni che possono aumentare o sopprimere citotossicità. Abbiamo poi esaminato i geni di lievito candidato nel nostro C. elegans alfa-sinucleina indotta saggio neurodegenerazione e convalidato correttamente molti di questi obiettivi 3, 4. La nostra metodologia prevede la generazione di C. elegans DA neurone specifico vettore di espressione tramite la clonazione di ricombinazione cDNA gene candidato sotto il controllo del Pdat-1 promotore. Questi plasmidi sono poi microiniettati nel wild-type (N2) vermi, insieme a un marcatore per la trasformazione di successo. Più linee transgeniche stabili produrre la proteina nei neuroni DA candidato si ottengono e poi attraversato in modo indipendente l&#39;alfa-sinucleina ceppo degenerative e valutati per la neurodegenerazione, sia l&#39;animale e il livello di singolo neurone, nel corso del tempo.

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

Questo video illustra come utilizzare C. elegans per valutare neurodegenerazione dei neuroni dopaminergici come modello per la malattia di Parkinson. Inoltre, gli schermi genetici sono usati per identificare i fattori che o migliorare la degenerazione o sono neuroprotettivi.

L&#39;applicazione di una degenerazione C. elegans Dopamina Saggio Neuron per la convalida dei potenziali morbo di Parkinson geni

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 Views.  University of Alabama. Questo video illustra come utilizzare C. elegans per valutare neurodegenerazione dei neuroni dopaminergici come modello per la malattia di Parkinson. Inoltre, gli schermi genetici sono usati per identificare i fattori che o migliorare la degenerazione o sono neuroprotettivi.

Video: 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: sviluppo di nuove terapie per la malattia di Parkinson

Ricerca

Biologia

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.

Generazione di Stabile transgenici C. elegans Utilizzando Microiniezione

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.

Modello murino per la malattia di Parkinson: da 6-OH lesione dopamina a test comportamentali

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.

Generazione di transgenici C. elegans Di Trasformazione Biolistic

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 produzione di C. elegans Transgeni attraverso recombineering con il GalK Marcatori selezionabili

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: Rilevamento delle alterazioni numeriche e strutturali in tutti i 24 cromosomi umani simultaneamente utilizzando un nuovo metodo di analisi FISH 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.

Utilizzo di RNA-mediata strategia di alimentazione di interferenza secondo schermo per geni coinvolti nella regolazione Dimensione del corpo nel nematode<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 Chemiotassi 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.

Un metodo per la coltura embrionale<em&gt; C. elegans</em&gt; Celle

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.

Misurare gli effetti dei batteri su<em&gt; C. Elegans</em&gt; Comportamento utilizzando un saggio di ritenzione dell&#39;uovo

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 ...