Queremos reconocer el espíritu de cooperación de todos los miembros Caldwell laboratorio. Movimiento de investigación trastornos en el laboratorio ha sido apoyado por la distonía Bachmann-Strauss y la Fundación Parkinson, Reino Parkinson Foundation, American Parkinson Disease Association, Asociación de la Enfermedad de Parkinson de Alabama, la Fundación Michael J. Fox para la Investigación del Parkinson, y una investigación de Pregrado

A. Construcción de plásmido de expresión Dos plásmidos son necesarios: una para la expresión específica de tejido del gen de interés y un segundo como marcador de transformación seleccionables (aunque el marcador plásmido está generalmente disponible dentro de la comunidad de investigación). Experimental plásmido <ol><li> Seleccione un promotor que se expresa en el tipo de tejidos / células de interés, en este caso, el transportador de DA (Pdat-1) se utiliza el promotor. Este plásmido de expresión fue creada como una puerta de enlace Invitrogen sistema compatible con vector destino (pDEST-DAT-1) para permitir la inserción de un gen de interés aguas abajo del promotor de la clonación de recombinación 1. </li><li> El cDNA del gen de interés es amplificado por PCR utilizando los cebadores que contienen la puerta de enlace att secuencia B de recombinación. El cDNA amplificado por primera vez se recombinan en el vector de donantes pDONR201 para crear el vector de entrada y luego se transformó en la cepa E. coli DH5a. Después de la selección de recombinantes éxito y mini-prep aislamiento de ADN, el vector de entrada es recombinada en el pDEST-DAT-1 vector de destino para crear el plásmido de expresión. Detalles sobre la clonación de recombinación están disponibles, ya sea el manual de Invitrogen Gateway o en Caldwell et al. 5. </li></ol> Marcador seleccionable plásmido Un marcador plásmido transgénico consiste en un vector con un promotor que determinan la expresión de una proteína fluorescente en un tejido obvio. En este procedimiento en particular, el promotor unc-54 unidades de expresión de la proteína de cerezo en el cuerpo de la pared muscular (punción-54:: cereza). B. Generación de transgénicos a través de C. elegans microinyección Ver artículo relacionado Jove: <a href="https://www.jove.com/index/details.stp?ID=833">https://www.jove.com/index/details.stp?ID=833</a> C. cruza genética para el análisis de la neurodegeneración DA <ol><li> Los gusanos son cultivados usando procedimientos estándar de 6. </li><li> Lugar 10 Pdat-1:: α-syn, Pdat-1:: GFP los hombres 1, 2 sobre una placa de acoplamiento con 4.3 L4 etapa hermafroditas transgénicos (que expresa Pdat-1:: gen X y punción de 54:: cherry) . Esto debe realizarse de forma individual para cada una de las tres líneas separadas estable creado. Después de dos días, eliminar a los machos. </li><li> Inspeccione la generación F1, si hay varios hijos varones, el apareamiento tuvo éxito. </li><li> Clon de 5 hermafrodita L4 animales de cada cruz que exhiben tanto el Pdat-1:: GFP fluorescentes marcador (heredado de los padres varones) y el 54 punción:: cherry pared fluorescentes cuerpo marcador de músculo (se hereda de los padres hermafrodita). Que los animales clonados tienen que estar en la etapa de L4 para asegurarse de que aún no han apareado con machos presentes en la placa. Les permitan auto-cross y producir una progenie F2. </li><li> F2 animales producidos en el paso 3 se clonan a cabo. En concreto, la transferencia de los animales ~ 5.10 que exhiben ambos marcadores fluorescentes para sus platos individuales. Pantalla de la generación F3 para las placas donde el 100% de la GFP animales expresan en las neuronas DA y algunos de los animales se expresa la cereza en las células del cuerpo músculo de la pared. Estos animales serán homocigotos para el Pdat-1:: α-syn, Pdat-1:: gen GFP, mientras que de forma estable la transmisión de la recién creada transgén (gen X). </li></ol> D. Análisis de las neuronas dopaminérgicas <ol><li> Prepare un plato fresco para cada una de las tres líneas creadas anteriormente, así como la Pdat-1:: α-syn, Pdat-1:: GFP sola cepa. Les permiten crecer a la edad adulta. </li><li> La transferencia de los adultos 30-40 transgénicos de cada línea en un plato fresco. Les permitan sentar los embriones de 4 horas a 20 º C. </li><li> Retire los adultos. Permita que los embriones para desarrollar durante 3-4 días a 20 º C hasta que alcancen la etapa de L4. </li><li> Elige ~ 100 L4 transgénicos animales a una placa que contiene 0,04 mg / ml 5-fluoro-2&#39;-desoxiuridina (FUDR). Este desarrollo nucleótidos bloques analógicos de la próxima generación a través de la inhibición de la síntesis de ADN, evitando así la descendencia de los animales de experimentación abrumadora 7. Proteger las placas FUDR de la luz, ya que es sensible a la luz. </li><li> Los animales adultos se analizaron en varios días después de la puesta de huevos. El día de análisis apropiados se determinan empíricamente. Por ejemplo, hemos determinado que el 77%, 87% y 90% de los Pdat-1:: α-syn, Pdat-1:: GFP animales exhiben degenerado neuronas DA en los días 6, 7 y 10 de desarrollo posterior, respectivamente. Por lo tanto, si podemos predecir que el transgen de interés podría aumentar la neurodegeneración, vamos a analizar en el día 6 y / o 7. Del mismo modo, los transgenes que podrían suprimir la neurodegeneración se analizarán en los días 7 y 10. </li><li> Hacer 4 pastillas de dulce de agarosa (a diferencia de las pastillas de microinyección agar, estos se utilizan frescos y no permitir que se seque). Se establecen dos portaobjetos de microscopio que tienen un pedazo de cinta a través de ellas, la cinta sirve como separador de pastillas de equivalente de espesor. Ponga un terciodeslice entre ellos en el banco (que se colocan de tres al día). Coloque una gota de agarosa fundida en el centro de la diapositiva y rápidamente poner otra diapositiva en la parte superior de la agarosa, perpendicular y en las tres diapositivas. Hacer varias almohadillas adicionales, dejando a los dos portaobjetos, hasta que la pastilla se encuentra listo para su uso. </li><li> Para el análisis de gusano neuronas DA, el lugar una caída de 6 l de levamisol mm 3 (un anestésico) en un 22 x 30 mm cubierta de vidrio. Recoger 40 animales adultos (10 extra más allá de los 30 que anotó) dentro de la gota, y luego invertir la cubierta de cristal en la almohadilla de agarosa. Repita este procedimiento para cada una de las otras líneas. </li><li> Puntuación 30 animales por cada línea de la presencia de cada CEP y la neurona ADE utilizando un microscopio compuesto con epifluorescencia y un cubo de filtro que permite la visualización del FITC o GFP. Nosotros usamos un filtro de ganar a las buenas prácticas agrarias HyQ cubo (Chroma Technology). Anota los datos en la hoja de registro adjunta. Animales de tipo salvaje mantendrá fluorescencia de GFP completa en todos los CEP 4 y 2 neuronas ADE. Animales individuales mostrando la pérdida de neuronas DA se califican como no-WT o &quot;degeneración&quot;. Del mismo modo, el grado de degeneración se puede marcar, contando el total de las neuronas perdidas en cada animal, así como a la población. </li><li> Repetir el análisis dos veces más (30 animales más por línea de prueba). El número total de gusanos de tipo salvaje exhibiendo las neuronas DA de las tres rondas de análisis es el promedio. El análisis estadístico para la neuroprotección continuación, se realiza utilizando la t de Student-test (p &lt;0,05) para comparar los gusanos de control (Pdat-1:: α-syn, Pdat-1:: GFP) con las cepas que sobreexpresan genes en las neuronas 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 pérdida depende de la edad de las neuronas de dopamina es una característica clínica de la enfermedad de Parkinson y se ha asociado con la acumulación o el mal plegamiento de una proteína llamada alfa-sinucleína. Aquí se demuestra la forma de etiqueta, a través de un transgén fluorescentes, las neuronas dopaminérgicas de la C. elegans e imitar la neurodegeneración en la enfermedad de Parkinson por coexpressing humana alfa-sinucleína en estas células. Este video muestra la metodología para el crecimiento, cr...

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<td>Coverglass 20x30 mm</td>
<td>&nbsp;</td>
<td>Fisher</td>
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<td>Plain, 3x1"</td>
<td>Fisher</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

Mejoras en el diagnóstico y el tratamiento de la enfermedad de Parkinson (EP) dependen de los conocimientos sobre factores de vulnerabilidad que hacen que las poblaciones en riesgo. En el proceso de tratar de identificar nuevos factores genéticos asociados con la enfermedad, los científicos han generado muchas listas de genes candidatos, los polimorfismos y proteínas que representan importantes avances, pero estos conductores siguen mecánicamente indefinido. Nuestro trabajo está dirigido a reducir significativamente estas listas mediante la explotación de las ventajas de un sistema modelo animal simple. Mientras que los humanos tienen miles de millones de neuronas, la lombriz Caenorhabditis elegans microscópica tiene precisamente 302, de los cuales sólo ocho producir dopamina (DA) en hemaphrodites. Expresión de un gen humano que codifica la proteína asociada a la EP, la alfa-sinucleína, en C. elegans resultados neuronas DA de la dosis y la neurodegeneración dependiente de la edad. Gusanos expresar humana alfa-sinucleína en las neuronas DA son isogénicas y expresar tanto las buenas prácticas agrarias y los derechos humanos alfa-sinucleína en el promotor del transportador de DA (Pdat-1). La presencia de GFP sirve como un marcador visualiza fácilmente para el seguimiento de la neurodegeneración DA en estos animales. Inicialmente se demostró que la alfa-sinucleína la neurodegeneración inducida por DA podrían ser rescatados en estos animales por torsinA, una proteína con actividad chaperona molecular 1. Además, el candidato PD relacionadas con los genes identificados en nuestro laboratorio a través de grandes esfuerzos de detección RNAi mediante una prueba de mal plegamiento de la alfa-sinucleína eran entonces sobre-expresado en C. elegans neuronas DA. Se determinó que cinco de los siete genes probados representado moduladores candidato importante de la EP, ya que rescató a la alfa-sinucleína inducida por la neurodegeneración DA 2. Además, el laboratorio de Lindquist (este número de Jove) ha llevado a cabo las pantallas de la levadura por el que la alfa-sinucleína toxicidad dependiente se utiliza como una lectura de los genes que pueden aumentar o suprimir la citotoxicidad. Nos examinó a continuación los genes candidatos de la levadura en nuestro ensayo de C. elegans neurodegeneración alfa-sinucleína y la inducida por validado con éxito muchos de estos tres objetivos, 4. Nuestra metodología se basa en la generación de un vector de expresión de C. elegans DA neuronal específica con la clonación de cDNAs de recombinación de genes candidatos bajo el control del Pdat-1 promotor. Estos plásmidos son entonces microinyección de tipo salvaje (N2) gusanos, junto con un marcador de selección para la transformación exitosa. Múltiples líneas transgénicas estables de producir la proteína en las neuronas DA candidato se obtienen y se cruzó de manera independiente en la cepa de la alfa-sinucleína degenerativas y evaluados por la neurodegeneración, en el animal y el nivel de las neuronas individuales, en el curso del envejecimiento.

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

Este video muestra cómo utilizar C. elegans para evaluar la neurodegeneración nerviosas dopaminérgicas como un modelo para la enfermedad de Parkinson. Además, las pantallas de genética se utilizan para definir los factores que mejoran la degeneración o neuroprotectoras.

Aplicación de un ensayo de C. elegans la degeneración neuronal de dopamina para la validación del potencial de la enfermedad de 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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Research

JoVE Journal

Biology

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

15.9K vistas.  University of Alabama. Este video muestra cómo utilizar C. elegans para evaluar la neurodegeneración nerviosas dopaminérgicas como un modelo para la enfermedad de Parkinson. Además, las pantallas de genética se utilizan para definir los factores que mejoran la degeneración o neuroprotectoras.

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: desarrollo de nuevas terapias para la enfermedad de Parkinson

Investigación

Biología

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.

Generación de transgénicos estable C. elegans con microinyección

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.

Modelo murino de enfermedad de Parkinson: de la lesión 6-OH dopamina a prueba de comportamiento

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.

Generación de transgénicos C. elegans Por la transformación biolística

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 producción de C. elegans Los transgenes a través de recombinería con la GALK Marcador seleccionable

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: Detección de alteraciones numéricas y estructurales en todos los 24 cromosomas humanos de manera simultánea utilizando un nuevo ensayo 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.

El uso de ARN de interferencia mediada estrategia de alimentación para la detección de genes implicados en la regulación del cuerpo Tamaño del nematodo<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 Ensayo de quimiotaxis

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 método para cultivar embrionarias<em&gt; C. elegans</em&gt; Células

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.

Medición de los efectos de las bacterias en<em&gt; C. Elegans</em&gt; Comportamiento El uso de un ensayo de retención de huevo

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 ...