Nous tenons à remercier le Dr Michael Baker pour son aide dans la génération des constructions pour le gène knock-in et de fournir les films en accéléré de la croissance des projections sensorielles. Nous tenons également à remercier la Virginie Vandelinder d&#39;assistance technique avec les expériences pistolet pneumatique.

Les particules peuvent être enduits de réactifs différents, tels que les molécules ARN double brin, l&#39;ADN des plasmides ou des colorants. Le type et le diamètre des particules sont choisies selon le réactif et la profondeur des cellules cibles dans le tissu: les grosses particules pénètrent plus loin, mais peut causer des dommages aux tissus. 1. Préparation de particules de revêtement ARNdb <ol><li> Placer 100% d&#39;isopropanol sur la glace. </li><li> Resuspendre 5 mg de particules d&#39;or (S1600ri, Seashell Technology, LLC; diamètre moyen 1,6 um) dans 100 ml de tampon de liaison. Lorsque la solution est prête, passez à l&#39;étape suivante. </li><li> Diluer la solution de particules en ajoutant 100 ul de tampon de liaison, brièvement au vortex et sonifier pendant 1-2 min afin d&#39;éviter l&#39;agglutination. </li><li> Ajouter 5-10 pg d&#39;ARN double brin / siRNA (dsRNA / siARN doit être dissous dans l&#39;eau à une concentration de ~ 1μg/μl). </li><li> Vortex et laisser à température ambiante pendant 2 min. Ces résultats intervalle de concentration et le temps de siARN saturé de particules d&#39;or. </li><li> Pellet les complexes de particules siARN / or par centrifugation à 2500 rpm ~ dans une centrifugeuse de paillasse pour ~ 15 sec. </li><li> Retirer délicatement le surnageant et ajouter 750 ml d&#39;isopropanol froid avec une perturbation minimale de la pastille. Spin brièvement et éliminer le surnageant. </li><li> Resuspendre les particules d&#39;or dans 100 ul froid 100% d&#39;isopropanol, puis soniquer brièvement dans un bain d&#39;ultrasons (2-3 impulsions) pour briser les agglomérats de particules. Enfin, versez les particules en suspension sur une lame de verre et laisser sécher à température ambiante. </li></ol> 2. Préparer le montage expérimental pour un dosage spécifique <ol><li> Chaque arme a un diamètre de buse différents ouverture, allant d&#39;un minimum de 50 microns ~ à ~ 3mm. L&#39;spécifiques à utiliser pour le pistolet est donc choisi en fonction de la surface du tissu d&#39;être ciblés. </li><li> La pression, il est ajusté à la profondeur dans les tissus des cellules cibles, généralement jusqu&#39;à 100 microns ~ (pressions plus élevées peuvent livrer les particules plus profond). La distance entre l&#39;embryon et l&#39;extrémité de la buse affecte également la profondeur de pénétration, comme l&#39;or particules essoufflement dans l&#39;air. </li></ol> 3. Chargement du pistolet avec les particules pré-enduites Particules devrait être chargé comme une poudre sèche dans les lignes d&#39;injection tube Tygon particules reliée au collecteur. Ce tube, qui devraient être conservés dans une atmosphère sèche à 4 ° C lorsqu&#39;il n&#39;est pas utilisé, peut être ré-utilisés dans des expériences supplémentaires avec les mêmes réactifs. Notre dispositif expérimental peut fournir jusqu&#39;à deux réactifs différents par test par le biais des ports distincts dans le collecteur. <ol><li> Gratter les particules séchées revêtement du verre diapositives en utilisant le bord d&#39;une lamelle de verre ou une lame de rasoir avant de les charger dans la ligne de tube Tygon. Ce tube Tygon seront spécifiques seulement à ce réactif pour éviter la contamination croisée. </li><li> Comme vous chargez les particules, plier le tube en forme de U près du connecteur, pour maintenir les particules de se propager le long du tube entier et concentrées à proximité du connecteur. Ramassez les particules avec une petite spatule ou d&#39;un morceau de papier plié pèse, et la charge environ la moitié d&#39;entre eux dans les tubes de chaque série d&#39;embryons. Appuyez sur le tube pendant l&#39;étape de chargement de répartir les particules uniformément. </li></ol> 4. Préparation des animaux Avant et après la livraison des particules d&#39;or, les embryons sont conservés dans l&#39;eau d&#39;étang artificiel stérile à température ambiante. <ol><li> Placer les embryons, jusqu&#39;à la face ventrale, dans une rainure taillée dans un plat, 5mm d&#39;épaisseur morceau de caoutchouc de silicone (Sylgard 184, Dow Corning, Midland, MI) et les anesthésier dans une solution d&#39;éthanol à 8% dans l&#39;eau d&#39;étang artificiel stérile ( jeunes embryons à des stades E6-E8 peut rester dans l&#39;eau des étangs artificiels, sans éthanol à travers l&#39;expérience). </li><li> Immédiatement avant le tournage des particules, d&#39;abaisser le niveau de bain de découvrir la face ventrale de l&#39;embryon en enlevant une partie de la solution. Placer un morceau de papier de soie avec un petit trou coupé au milieu sur le dessus de celui-ci afin de le stabiliser et de maintenir sa surface de séchage. </li></ol> 5. La livraison des particules enrobées dans le tissu cible Une charge de particules peuvent généralement être utilisés pour un maximum de dix coups, avec un seul tir en général la livraison de l&#39;ordre de quelques centaines de particules. <ol><li> Générer un coup en ouvrant l&#39;une des soupapes de 0,3 s, ce qui provoque l&#39;injection d&#39;un bolus de particules provenant de la ligne correspondante tuyau dans le pistolet. </li><li> Appuyez sur le tube doucement entre les coups de feu pour déloger les particules de la paroi du tube et donc de faciliter leur injection dans le flux, il de l&#39;arme. </li><li> Pour couvrir la surface du tissu d&#39;intérêt, des coups multiples peuvent être nécessaires. </li><li> Après la livraison des particules, placer les embryons de nouveau dans l&#39;eau des étangs artificiels, de préférence un seul embryon par puits dans une plaque multi-puits. </li></ol> 6. Immunocoloration et l&#39;étiquetage des neurones Gardez les embryons à température ambiante et dans l&#39;obscurité pendant 1 à 3 jours après la livraison des particules, jusqu&#39;à ce que l&#39;ARNi (ou l&#39;expression ectopique) est atteint. Nous avons ensuite effectuer l&#39;une des procédures suivantes sur les échantillons expérimentaux et contrôles: <ol><li> L&#39;hybridation in situ d&#39;analyse pour la présence ou l&#39;absence de l&#39;ARNm de la nétrine. Marquée à la digoxigénine sangsue ribosondes nétrine sont hybridées à des embryons de sangsue ensemble. </li><li> Immunocytochimie au dosage de la protéine nétrine et d&#39;autres protéines qui sont uniques aux tissus neuronaux (comme la tubuline acétylée anti). La procédure est effectuée également sur toute monture embryons. </li><li> Microinjections de colorants lipophiles (DII et DIO) en neurones unique, pour comble de teinture pour étiqueter les tonnelles complète des neurones d&#39;intérêt (de pression neurones mécano dans notre cas) dans des segments traitées et les témoins. Ce dosage est effectué sur les embryons vivants (intacts ou disséqués). </li></ol>

<ol>
	<li>Baker, M. W., Macagno, E. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNAi+of+the+receptor+tyrosine+phosphatase+HmLAR2+in+a+single+cell+of+an+intact+leech+embryo+leads+to+growth-cone+collapse.">RNAi of the receptor tyrosine phosphatase HmLAR2 in a single cell of an intact leech embryo leads to growth-cone collapse.</a> Current Biology. 10, 1071-1074 (2000).</li><li>Baker, M. W., Macagno, E. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+Hirudo+medicinalis+DNA+Promoters+for+Targeted+Gene+Expression.">Characterization of Hirudo medicinalis DNA Promoters for Targeted Gene Expression.</a> Journal of Neuroscience Methods. 156, 145-153 (2006).</li><li>Gan, W. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+expression+of+a+leech+netrin+suggests+roles+in+the+formation+of+longitudinal+nerve+tracts+and+in+regional+innervation+of+peripheral+targets.">Cellular expression of a leech netrin suggests roles in the formation of longitudinal nerve tracts and in regional innervation of peripheral targets.</a> Journal of Neurobiology. 40, 103-115 (1999).</li><li>Juven-Gershon, T., Cheng, S., Kadonaga, J. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rational+design+of+a+super+core+promoter+that+enhances+gene+expression.">Rational design of a super core promoter that enhances gene expression.</a> Nature Methods. 3, 917-922 (2006).</li><li>Rinberg, D., Simonnet, C., Groisman, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pneumatic+capillary+gun+for+ballistic+delivery+of+microparticles.">Pneumatic capillary gun for ballistic delivery of microparticles.</a> Applied Physics Letters. 87, 014103 (2005).</li><li>Shefi, O., Simonnet, C., Baker, M. W., Glass, J. R., Macagno, E. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microtargeted+gene+silencing+and+ectopic+expression+in+live+embryos+using+biolistic+delivery+with+a+pneumatic+capillary+gun.">Microtargeted gene silencing and ectopic expression in live embryos using biolistic delivery with a pneumatic capillary gun.</a> Journal of Neuroscience. 26, 6119-6123 (2006).</li></ol>

Dans cette démonstration, nous avons utilisé le pistolet pneumatique capillaires pour fournir des particules d&#39;or dans les cellules localisées dans le volume des petits un embryon vivant sangsue à knock-in et knock-down gènes. La région cible ont généralement un diamètre de ~ 150 um et les particules ont été trouvés ~ 15 um autour de la profondeur de pénétration moyenne, qui était réglable entre 0 et 50 um. Lorsque les particules ont été recouvertes de molécules ARN double brin de l&#39;axone nétrine facteur de guida...

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<td>gold particles</td>
<td>Other</td>
<td>Seashell Technology, LLC</td>
<td>S1600ri</td>
<td>&nbsp;</td>
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<td>binding buffer</td>
<td>Reagent</td>
<td>Seashell Technology, LLC</td>
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<td>silicone rubber </td>
<td>Reagent</td>
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localized rnai and ectopic gene expression in the medicinal leech

Dans cette vidéo, nous montrons l&#39;utilisation d&#39;un pistolet pneumatique capillaires pour la livraison précise biolistique des réactifs dans les tissus vivants. Nous utilisons la procédure à des modèles de perturber l&#39;expression des gènes dans des segments choisis d&#39;embryons de sangsue, laissant les segments non traitées comme des contrôles internes. Le pistolet pneumatique capillaire peut être utilisée pour atteindre les couches internes de cellules à des stades précoces du développement, sans ouvrir le spécimen. En tant que méthode pour l&#39;introduction de substances dans localisée des tissus vivants, de la délivrance biolistique avec le pistolet a plusieurs avantages: il est rapide, sans contact et non destructive. En outre, une seule arme capillaire peut être utilisée pour la livraison indépendant de différentes substances. La région de livraison peuvent avoir des dimensions latérales de ~ 50 à 150 um et s&#39;étend sur environ 15 um autour de la profondeur de pénétration moyenne, qui est réglable entre 0 et 50 um. Cette livraison a l&#39;avantage d&#39;être en mesure de cibler un nombre limité de cellules dans un endroit choisi intermédiaire entre cellule unique renverser par microinjection et démontable par des injections systémiques extracellulaire ou par des approches génétiques. Pour faire tomber ou de frapper dans l&#39;expression de la nétrine axone molécule orientation, qui est naturellement exprimé par certains neurones centraux et dans la paroi ventrale du corps, mais pas le domaine dorsale, nous délivrer des molécules d&#39;ARN double brin ou ADN plasmidique dans la paroi du corps et ganglions centraux. Cette procédure comprend les étapes suivantes: (i) la préparation du dispositif expérimental pour un dosage précis (ajustement de la pression d&#39;accélération), (ii) l&#39;enrobage des particules avec des molécules d&#39;ARN double brin ou d&#39;ADN, (iii) de chargement des particules enrobées dans le pistolet, jusqu&#39;à deux réactifs dans un essai, (iv) la préparation des animaux pour la livraison des particules, (v) la livraison de particules enrobées dans le tissu cible (paroi du corps ou des ganglions), et (vi) le traitement des embryons (immunomarquage, l&#39;immunohistochimie et neuronale étiquetage) pour visualiser les résultats, généralement de 2 à 3 jours après la livraison. Lorsque les particules ont été revêtus de la nétrine ARNdb, ils ont causé bien visible le knock-down de l&#39;expression nétrine que seulement eu lieu dans des cellules contenant des particules (généralement, 1-2 particules par cellule). Particules enrobées avec un encodage de fluorescence EGFP plasmide induite dans les cellules neuronales quand ils ont cessé dans leurs noyaux.

Watch this Scientific Journal Video about Localized RNAi and Ectopic Gene Expression in the Medicinal Leech at JoVE.com

Localized RNAi and Ectopic Gene Expression in the Medicinal Leech

Dans cette vidéo, nous montrons une procédure pour une livraison précise biolistique des réactifs dans le tissu vivre avec un canon à gènes miniatures roman. Nous sommes renversant l&#39;expression de la nétrine axone molécule conseils dans les embryons de sangsue en fournissant des molécules d&#39;ARN double brin dans le mur ventrale du corps et les ganglions des segments unique.

Localisé ARNi et l&#39;expression des gènes extra-utérine dans la sangsue médicinale

In this video, we show the use of a pneumatic capillary gun for the accurate biolistic delivery of reagents into live tissue. We use the procedure to ...

localized-rnai-and-ectopic-gene-expression-in-the-medicinal-leech

Research

JoVE Journal

Biology

9.5K vues.  University of California San Diego - UCSD. Dans cette vidéo, nous montrons une procédure pour une livraison précise biolistique des réactifs dans le tissu vivre avec un canon à gènes miniatures roman. Nous sommes renversant l'expression de la nétrine axone molécule conseils dans les embryons de sangsue en fournissant des molécules d'ARN double brin dans le mur ventrale du corps et les ganglions des segments unique.

Vidéo: Localized RNAi and Ectopic Gene Expression in the Medicinal Leech

The Preparation of Primary Hematopoietic Cell Cultures From Murine Bone Marrow for Electroporation

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser MXcell  electroporation system and Gene Pulser  electroporation buffer were specifically developed to transfect nucleic acids into mammalian cells and difficult-to-transfect cells, such as primary and stem cells.This video demonstrates how to establish primary hematopoietic cell cultures from murine bone marrow, and then prepare them for electroporation in the MXcell system. We begin by isolating femur and tibia. Bone marrow from both femur and tibia are then harvested and cultures are established. Cultured bone marrow cells are then transfected and analyzed.

This procedure describes how to establish primary hematopoietic cell cultures from murine bone marrow and is followed by transfection using the Gene Pulser MXCell electroporation system.

La préparation des cultures primaires de cellules hématopoïétiques de la moelle osseuse murine pour l&#39;électroporation

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser ...

Recherche

Biologie

Spinal Cord Electrophysiology

The neonatal mouse spinal cord is a model for studying the development of neural circuitries and locomotor movement. We demonstrate the spinal cord dissection and preparation of recording bath artificial cerebrospinal fluid used for locomotor studies. Once dissected, the spinal cord ventral nerve roots can be attached to a recording electrode to record the electrophysiologic signals of the central pattern generating circuitry within the lumbar cord.

A demonstration of the isolation of neonatal mouse spinal cord for electrophysiologic studies.

Spinal Cord électrophysiologie

The neonatal mouse spinal cord is a model for studying the development of neural circuitries and locomotor movement. We demonstrate the spinal cord ...

Using the Gene Pulser MXcell Electroporation System to Transfect Primary Cells with High Efficiency

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser MXcell electroporation system and Gene Pulser electroporation buffer (Bio-Rad) were specifically developed to easily transfect nucleic acids into mammalian cells and difficult-to-transfect cells, such as primary and stem cells. We will demonstrate how to perform a simple experiment to quickly identify the best electroporation conditions. We will demonstrate how to run several samples through a range of electroporation conditions so that an experiment can be conducted at the same time as optimization is performed. We will also show how optimal conditions identified using 96-well electroporation plates can be used with standard electroporation cuvettes, facilitating the switch from electroporation plates to electroporation cuvettes while maintaining the same electroporation efficiency. In the video, we will also discuss some of the key factors that can lead to the success or failure of electroporation experiments.

This procedure shows how to use the Gene Pulser MXcell electroporation system to rapidly and easily identify the best electroporation conditions for mouse embryonic fibroblasts (MEFs) or other primary cells. Considerations for troubleshooting are also discussed in the associated video.

Utilisation du système d&#39;électroporation Gene Pulser MXcell à transfecter des cellules primaires à haut rendement

Robotics and Dynamic Image Analysis for Studies of Gene Expression in Plant Tissues

Gene expression in plant tissues is typically studied by destructive extraction of compounds from plant tissues for in vitro analyses. The methods presented here utilize the green fluorescent protein (gfp) gene for continual monitoring of gene expression in the same pieces of tissues, over time. The gfp gene was placed under regulatory control of different promoters and introduced into lima bean cotyledonary tissues via particle bombardment. Cotyledons were then placed on a robotic image collection system, which consisted of a fluorescence dissecting microscope with a digital camera and a 2-dimensional robotics platform custom-designed to allow secure attachment of culture dishes. Images were collected from cotyledonary tissues every hour for 100 hours to generate expression profiles for each promoter. Each collected series of 100 images was first subjected to manual image alignment using ImageReady to make certain that GFP-expressing foci were consistently retained within selected fields of analysis. Specific regions of the series measuring 300 x 400 pixels, were then selected for further analysis to provide GFP Intensity measurements using ImageJ software. Batch images were separated into the red, green and blue channels and GFP-expressing areas were identified using the threshold feature of ImageJ. After subtracting the background fluorescence (subtraction of gray values of non-expressing pixels from every pixel) in the respective red and green channels, GFP intensity was calculated by multiplying the mean grayscale value per pixel by the total number of GFP-expressing pixels in each channel, and then adding those values for both the red and green channels. GFP Intensity values were collected for all 100 time points to yield expression profiles. Variations in GFP expression profiles resulted from differences in factors such as promoter strength, presence of a silencing suppressor, or nature of the promoter. In addition to quantification of GFP intensity, the image series were also used to generate time-lapse animations using ImageReady. Time-lapse animations revealed that the clear majority of cells displayed a relatively rapid increase in GFP expression, followed by a slow decline. Some cells occasionally displayed a sudden loss of fluorescence, which may be associated with rapid cell death. Apparent transport of GFP across the membrane and cell wall to adjacent cells was also observed. Time lapse animations provided additional information that could not otherwise be obtained using GFP Intensity profiles or single time point image collections.

We report a method for introduction, tracking and quantitative analysis of GFP expression in plant cells. This method utilizes a custom-designed robotics system for semi-continuous image collection from large numbers of samples, over time. We also demonstrate the use of ImageJ and ImageReady for analysis of image series.

Robotique et d&#39;analyse d&#39;images dynamiques pour les études d&#39;expression génique dans les tissus végétaux

Gene expression in plant tissues is typically studied by destructive extraction of compounds from plant tissues for in vitro analyses. The methods ...

Using an Automated Cell Counter to Simplify Gene Expression Studies: siRNA Knockdown of IL-4 Dependent Gene Expression in Namalwa Cells

The use of siRNA mediated gene knockdown is continuing to be an important tool in studies of gene expression.  siRNA studies are being conducted not only to study the effects of downregulating single genes, but also to interrogate signaling pathways and other complex interaction networks.  These pathway analyses require both the use of relevant cellular models and methods that cause less perturbation to the cellular physiology.  Electroporation is increasingly being used as an effective way to introduce siRNA and other nucleic acids into difficult to transfect cell lines and primary cells without altering the signaling pathway under investigation. There are multiple critical steps to a successful siRNA experiment, and there are ways to simplify the work while improving the data quality at several experimental stages.  To help you get started with your siRNA mediated gene knockdown project, we will demonstrate how to perform a pathway study complete from collecting and counting the cells prior to electroporation through post transfection real-time PCR gene expression analysis.  The following study investigates the role of the transcriptional activator STAT6 in IL-4 dependent gene expression of CCL17 in a Burkitt lymphoma cell line (Namalwa).  The techniques demonstrated are useful for a wide range of siRNA-based experiments on both adherent and suspension cells.  We will also show how to streamline cell counting with the TC10 automated cell counter, how to electroporate multiple samples simultaneously using the MXcell electroporation system, and how to simultaneously assess RNA quality and quantity with the Experion automated electrophoresis system.

This procedure describes a quick and easy workflow to introduce siRNA into difficult to transfect cell lines and follow gene expression by real-time PCR. Use of an automated cell counter, multi-well electroporation plate, and automated electrophoresis station provide quick and reliable results without the need for expensive robotic handling.

Aide d&#39;un compteur de cellules automatisé pour simplifier les études d&#39;expression génique: Knockdown siRNA de l&#39;IL-4 dans l&#39;expression des gènes dépendant cellules Namalwa

The use of siRNA mediated gene knockdown is continuing to be an important tool in studies of gene expression.  siRNA studies are being conducted not only ...

Paraffin-Embedded and Frozen Sections of Drosophila Adult Muscles

The molecular characterization of muscular dystrophies and myopathies in humans has revealed the complexity of muscle disease and genetic analysis of muscle specification, formation and function in model systems has provided valuable insight into muscle physiology. Therefore, identifying and characterizing molecular mechanisms that underlie muscle damage is critical. The structure of adult Drosophila multi-fiber muscles resemble vertebrate striated muscles 1 and the genetic tractability of Drosophila has made it a great system to analyze dystrophic muscle morphology and characterize the processes affecting muscular function in ageing adult flies 2. Here we present the histological technique for preparing paraffin-embedded and frozen sections of Drosophila thoracic muscles. These preparations allow for the tissue to be stained with classical histological stains and labeled with protein detecting dyes, and specifically cryosections are ideal for immunohistochemical detection of proteins in intact muscles. This allows for analysis of muscle tissue structure, identification of morphological defects, and detection of the expression pattern for muscle/neuron-specific proteins in Drosophila adult muscles. These techniques can also be slightly modified for sectioning of other body parts.

Paraffin-Embedded and Frozen Sections of Drosophila Adult Muscles

Identification of mechanisms underlying muscle damage is crucial. Here we present the histological technique for preparing paraffin-embedded and frozen sections of Drosophila thoracic muscles. This allows analysis of muscle morphology and localization of protein and other muscle cell components.

Les articles inclus en paraffine et congelés de Drosophile</emMuscles adultes>

The molecular characterization of muscular dystrophies and myopathies in humans has revealed the complexity of muscle disease and genetic analysis of ...

In vitro Reconstitution of the Active T. castaneum Telomerase

Efforts to isolate the catalytic subunit of telomerase, TERT, in sufficient quantities for structural studies, have been met with limited success for more than a decade. Here, we present methods for the isolation of the recombinant Tribolium castaneum TERT (TcTERT) and the reconstitution of the active T. castaneum telomerase ribonucleoprotein (RNP) complex in vitro.
Telomerase is a specialized reverse transcriptase1 that adds short DNA repeats, called telomeres, to the 3' end of linear chromosomes2 that serve to protect them from end-to-end fusion and degradation. Following DNA replication, a short segment is lost at the end of the chromosome3 and without telomerase, cells continue dividing until eventually reaching their Hayflick Limit4. Additionally, telomerase is dormant in most somatic cells5 in adults, but is active in cancer cells6 where it promotes cell immortality7.
The minimal telomerase enzyme consists of two core components: the protein subunit (TERT), which comprises the catalytic subunit of the enzyme and an integral RNA component (TER), which contains the template TERT uses to synthesize telomeres8,9. Prior to 2008, only structures for individual telomerase domains had been solved10,11. A major breakthrough in this field came from the determination of the crystal structure of the active12, catalytic subunit of T. castaneum telomerase, TcTERT1.
Here, we present methods for producing large quantities of the active, soluble TcTERT for structural and biochemical studies, and the reconstitution of the telomerase RNP complex in vitro for telomerase activity assays. An overview of the experimental methods used is shown in Figure 1.

In vitro Reconstitution of the Active T. castaneum Telomerase

Efforts to isolate the catalytic subunit of telomerase, TERT, in sufficient quantities for structural studies, have been met with limited success for more than a decade. Here, we present methods for the isolation of the recombinant Tribolium castaneum TERT (TcTERT) and the reconstitution of the active T. castaneum telomerase ribonucleoprotein (RNP) complex in vitro.

La reconstitution in vitro de l&#39;actif T. La télomérase castaneum

Efforts to isolate the catalytic subunit of telomerase, TERT, in sufficient quantities for structural studies, have been met with limited success for more ...

RNAi Mediated Gene Knockdown and Transgenesis by Microinjection in the Necromenic Nematode Pristionchus pacificus

Although it is increasingly affordable for emerging model organisms to obtain completely sequenced genomes, further in-depth gene function and expression analyses by RNA interference and stable transgenesis remain limited in many species due to the particular anatomy and molecular cellular biology of the organism. For example, outside of the crown group Caenorhabditis that includes Caenorhabditis elegans3, stably transmitted transgenic lines in non-Caenorhabditis species have not been reported in this specious phylum (Nematoda), with the exception of Strongyloides stercoralis4 and Pristionchus pacificus5. To facilitate the expanding role of P. pacificus in the study of development, evolution, and behavior6-7, we describe here the current methods to use microinjection for making transgenic animals and gene knock down by RNAi. Like the gonads of C. elegans and most other nematodes, the gonads of P. pacificus is syncitial and capable of incorporating DNA and RNA into the oocytes when delivered by direct microinjection. Unlike C. elegans however, stable transgene inheritance and somatic expression in P. pacificus requires the addition of self genomic DNA digested with endonucleases complementary to the ends of target transgenes and coinjection markers5. The addition of carrier genomic DNA is similar to the requirement for transgene expression in Strongyloides stercoralis4 and in the germ cells of C. elegans. However, it is not clear if the specific requirement for the animals' own genomic DNA is because P. pacificus soma is very efficient at silencing non-complex multi-copy genes or that extrachromosomal arrays in P. pacificus require genomic sequences for proper kinetochore assembly during mitosis. The ventral migration of the two-armed (didelphic) gonads in hermaphrodites further complicates the ability to inject both gonads in individual worms8. We also demonstrate the use of microinjection to knockdown a dominant mutant (roller,tu92) by injecting double-stranded RNA (dsRNA) into the gonads to obtain non-rolling F1 progeny. Unlike C. elegans, but like most other nematodes, P. pacificus PS312 is not receptive to systemic RNAi via feeding and soaking and therefore dsRNA must be administered by microinjection into the syncitial gonads. In this current study, we hope to describe the microinjection process needed to transform a Ppa-egl-4 promoter::GFP fusion reporter and knockdown a dominant roller prl-1 (tu92) mutant in a visually informative protocol.

RNAi Mediated Gene Knockdown and Transgenesis by Microinjection in the Necromenic Nematode Pristionchus pacificus

In model organisms, transgenesis can manipulate gene functions while RNAi can knockdown specific mRNA transcripts 1-2. This protocol aims to illustrate the techniques needed to introduce stably transmitted DNA and transient double stranded RNA into the necromenic nematode Pristionchus pacificus for studies in evolutionary, developmental, and behavioral biology.

ARNi Mediated inactivation génique et la transgénèse par microinjection dans le nématode Necromenic Pristionchus pacificus</em

Although it is increasingly affordable for emerging model organisms to obtain completely sequenced genomes, further in-depth gene function and expression ...

Analysis of Global RNA Synthesis at the Single Cell Level following Hypoxia

Hypoxia or lowering of the oxygen availability is involved in many physiological and pathological processes. At the molecular level, cells initiate a particular transcriptional program in order to mount an appropriate and coordinated cellular response. The cell possesses several oxygen sensor enzymes that require molecular oxygen as cofactor for their activity. These range from prolyl-hydroxylases to histone demethylases. The majority of studies analyzing cellular responses to hypoxia are based on cellular populations and average studies, and as such single cell analysis of hypoxic cells are seldom performed. Here we describe a method of analysis of global RNA synthesis at the single cell level in hypoxia by using Click-iT RNA imaging kits in an oxygen controlled workstation, followed by microscopy analysis and quantification.&nbsp; Using cancer cells exposed to hypoxia for different lengths of time, RNA is labeled and measured in each cell. This analysis allows the visualization of temporal and cell-to-cell changes in global RNA synthesis following hypoxic stress.

We describe a technique for analysis of global RNA synthesis in hypoxia using imaging. Click-chemistry labeling of RNA has not previously been performed under hypoxia and allows visualization of global RNA changes at the single cell level. This approach complements the existing averaged RNA techniques, allowing direct visualization of cell-to-cell changes in global RNA synthesis.

Analyse de Global ARN de synthèse à la Cellule Plain suivante hypoxie

Hypoxia or lowering of the oxygen availability is involved in many physiological and pathological processes. At the molecular level, cells initiate a ...

Measurement of Metabolic Rate in Drosophila using Respirometry

Metabolic disorders are a frequent problem affecting human health. Therefore, understanding the mechanisms that regulate metabolism is a crucial scientific task. Many disease causing genes in humans have a fly homologue, making Drosophila a good model to study signaling pathways involved in the development of different disorders. Additionally, the tractability of Drosophila simplifies genetic screens to aid in identifying novel therapeutic targets that may regulate metabolism. In order to perform such a screen a simple and fast method to identify changes in the metabolic state of flies is necessary. In general, carbon dioxide production is a good indicator of substrate oxidation and energy expenditure providing information about metabolic state. In this protocol we introduce a simple method to measure CO2 output from flies. This technique can potentially aid in the identification of genetic perturbations affecting metabolic rate.

Measurement of Metabolic Rate in Drosophila using Respirometry

Metabolic disorders are among one of the most common diseases in humans. The genetically tractable model organism D. melanogaster can be used to identify novel genes that regulate metabolism. This paper describes a relatively simple method which allows studying the metabolic rate in flies by measuring their CO2 production.