Nous remercions Chunxin Wang pour construire pTSO2:: GUS et pour des photos dans la figure 1, Detlef Weigel pour le partage du protocole de GUS-infaillible, Paja Sijacic, et Courtney Hollender pour leurs précieux commentaires. La recherche en laboratoire, Z. L s est soutenue par la National Science Foundation américaine (IOB0616096 et MCB0744752). ZL est partiellement pris en charge par l&#39;Université du Maryland de la station expérimentale agricole.

I. Floral-pendage de transformation d&#39;Arabidopsis thaliana <ol><li> Environ un mois avant la transformation, semer des graines d&#39;Arabidopsis sur 4 à 8 pots (5 pouces pots carrés) avec environ 10-15 plantes par pot. Pour les plantes à la fertilité normale, 4 pots de plantes par construire le plasmide sera suffisant. Si on a de transformer Arabidopsis mutantes avec une fertilité réduite, augmenter le nombre de pots en conséquence. </li><li> Les plantes sont cultivées dans des conditions standard (16 h de lumière et 8 h obscurité à 22 ° C). Si les plantes sont cultivées sous journée plus courte ou plus basse température, ils vont prendre plus de fleurs et plus de temps pour être prêt pour la transformation. Soins des plantes de bonne volonté d&#39;assurer des plantes saines, qui sont essentielles pour la transformation réussie. </li><li> Lorsque les plantes ont simplement boulonnés et commencé à fleurir. Ils sont maintenant prêts pour la transformation. Pour accroître l&#39;efficacité de transformation, 3-4 jours avant la transformation, coupez les pousses inflorescence principale dès qu&#39;ils ont boulonné à encourager davantage la formation de pousses secondaires. Arrosez les plantes le jour avant la transformation. Cela garantit que le sol ne sera pas s&#39;imprégner de l&#39;infiltration des médias trop pendant floral-trempage. </li><li> Deux jours avant la transformation, inoculer LB 40ml contenant des antibiotiques appropriés (dans le cas de pTSO2:: GUS, 50 pg / ml de kanamycine) par Agrobacterium tumefaciens souche GV3101 portant le pTSO2:: GUS construire. Le pTSO2:: GUS est cloné dans le vecteur qui possède pBIN20 gène NPTII qui confère une résistance à la kanamycine 3. Cultivez la nuit dans un incubateur agitateur à 28-30 ° C. </li><li> Le jour suivant, 8ml transfert de la culture de la nuit dans 400ml de milieu LB contenant 50 pg / ml de kanamycine. Cultivez la nuit dans un incubateur agitateur à 28-30 ° C. </li><li> Le jour de la transformation, lorsque la DO 600 de la culture d&#39;Agrobacterium atteint environ 0,8 (DO 600 de 0,5 à1 sont acceptables), ralentit la culture d&#39;Agrobacterium nuit à 8 minutes à 5000rpm. </li><li> Reprendre le culot dans 1 L d&#39;Agrobacterium (litre) des médias d&#39;infiltration (10 mM MgCl 2, 5% de saccharose, 0,44 mm 6-benzyladénine (BA), 0,3% Silwet L-77, la solution de 1x Gamborg s vitamines et l&#39;eau à l&#39;autoclave). Le milieu n&#39;a pas besoin d&#39;être autoclavés mais elle doit être préparée. Verser le milieu 1 L infiltration d&#39;Agrobacterium contenant dans un plat (comme 8 &quot;x15&quot; x2 &quot;Pyrex plat). </li><li> Inverser le pot (plantes ne tombent pas) et trempette toutes les parties aériennes des plantes dans la solution d&#39;infiltration, maintenez pendant 5 minutes un pot à la fois. Alors que toutes les parties aériennes de la plante sont immergées dans le milieu d&#39;infiltration, éviter de laisser le sol absorbent toute infiltration des médias afin de réduire les chances de croissance fongique sur le sol. </li><li> Après le trempage, placer tous les pots trempé de leur côté sur plusieurs couches de serviettes en papier dans un tiroir. Les serviettes en papier absorbent quantité d&#39;accès des médias d&#39;infiltration. Couvrir le bac avec une pellicule de plastique pour assurer une forte humidité. Placer le plateau dans la chambre de croissance des plantes. </li><li> Le lendemain, retirer la pellicule plastique et des serviettes en papier, et des pots lieu debout. Ne pas arroser ces plantes pendant quatre à cinq jours de plus. Ensuite, maintenir les plantes normalement jusqu&#39;à ce qu&#39;ils jeu graines et récolter les graines en vrac. </li><li> Verser dans les boîtes (150x15mm boîte de Petri) avec un milieu MS contenant 50 pg / ml de kanamycine (poids 2.2g de sel de Murashige et Skoog basale sans vitamine, dissoudre dans 500 ml d&#39;eau, le pH à 5,8 avec NaOH 1M, ajouter 4 grammes d&#39;agar, autoclave, frais et Ajouter à la kanamycine à 50 pg / ml en concentration finale). Les plaques sont conservés dans 4 ° C jusqu&#39;à ce que nécessaire. </li><li> Pour sélectionner des résistantes aux antibiotiques des plantes transgéniques, on a besoin de l&#39;écran 20 000 graines au minimum. 0,1 gramme est d&#39;environ 5000 graines. Poids et estimer la quantité de graines. </li><li> Stériliser les graines en les laver dans le tube Falcon de 15 ml d&#39;éthanol à 70% pendant 30 secondes. Laver les graines à l&#39;eau stérile une fois. Faire tremper les graines pendant 30 secondes dans une solution contenant de dilution 1:10 de l&#39;eau de Javel achetée en magasin de 5% (Clorox). Rincer les graines à l&#39;eau stérile trois à six fois. Dans hotte stérile, une pipette environ 5000 graines sur chaque EM (kanamycine) plaque, répandre les graines uniformément sur la plaque, les plaques d&#39;étanchéité avec 3M Micropore bande pour éviter la contamination. </li><li> Incuber les plaques à 4 ° C dans l&#39;obscurité pendant 3-4 jours, puis de transférer les plaques à une chambre (la chambre peut être même plante de croissance). Après environ 14 jours, les plants transgéniques restent vertes, mais celles non transgéniques sont en pâlissant et les mourants. Transfert des plants transgéniques sur le sol en ralentissant tirant les racines à partir de la moyenne et les a placés dans le sol. </li></ol> II. Examiner pTSO2:: profils d&#39;expression de GUS <ol><li> Récolte pTSO2:: GUS plants transgéniques et les placer dans de l&#39;acétone à 90% froid dans un flacon à scintillation en verre ou tubes de microcentrifugeusequi sont conservés sur la glace. Des plaques de microtitration en polystyrène (non polypropylène) peuvent également être utilisés si l&#39;on analyse un grand nombre d&#39;échantillons. </li><li> Après tous les échantillons sont récoltés, placer les flacons à température ambiante pendant 20 minutes. Pendant ce temps forment tampon de coloration douce sans X-gluc (0,2% de Triton X-100, 50mm NaHPO 4 Tampon pH 7,2, 2 mM ferrocyanure de potassium, potassium 2mM Ferricyanide) et placer sur la glace. </li><li> Enlever l&#39;acétone à partir des échantillons et ajouter le tampon de coloration. </li><li> Maquillage solution de coloration X-gluc (0,2% de Triton X-100, 50mm NaHPO 4 Tampon pH 7,2, 2 mM ferrocyanure de potassium, potassium 2mM Ferricyanide, 2mm x-gluc). Retirez le tampon de coloration à partir d&#39;échantillons et ajouter le tampon de coloration contenant X-gluc aux échantillons. </li><li> Infiltrez les échantillons sous vide pendant 15 à 20 minutes. Couper le vide lentement et vérifier que tous les échantillons couler sous la surface de la solution de coloration. Si nécessaire, répétez l&#39;infiltration jusqu&#39;à ce que tous les échantillons couler après que le vide est libéré. </li><li> Incuber les échantillons à 37 ° C la nuit dans le tampon de coloration avec X-gluc. </li><li> Retirer les échantillons de l&#39;incubateur et éliminer le tampon de coloration. Laver les échantillons successifs en série éthanol (20%, 35% et 50% d&#39;éthanol) à température ambiante pendant 30 minutes à chaque changement. </li><li> Fixer les tissus dans la FAA fixateur (50% d&#39;éthanol, le formaldéhyde à 5%, 10% d&#39;acide acétique, l&#39;eau reste) pendant 30 minutes à température ambiante. Retirez la FAA et ajouter 70% d&#39;éthanol. A ce moment les tissus peuvent être visualisées et photographié sous une loupe binoculaire équipée d&#39;un appareil photo numérique. Une couleur bleu foncé indique où TSO2 est exprimée (figure 1). Alternativement, les tissus peuvent être conservés à 4 ° C pour un visionnage ultérieur. <img src="/files/ftp_upload/1952/1952fig1.jpg" alt="Figure 1" /> Figure 1 pTSO2.:: Expression de GUS dans des plants transgéniques (A), les racines latérales (B), et l&#39;extrémité des racines (C). Notez que TSO2 promoteur est très actif dans les tissus jeunes et en divisant. Le profil d&#39;expression sporadiques montré en (C) est typique des gènes exprimés dans les phases spécifiques du cycle cellulaire. </li></ol> Les résultats représentatifs Une fois fait correctement, l&#39;efficacité de transformation devrait être d&#39;environ 0,1-0,2%. Dans un autre mot, on devrait obtenir de 50 à 10 plants transgéniques par un dépistage tous les 5000 graines. En moyenne, environ 100 lignées transgéniques peuvent être obtenus en transformant 4 pots de plantes de type sauvage. Pour les semis transgéniques contenant le pTSO2:: GUS construire 3, couleur bleu foncé qui reflète l&#39;activité GUS est trouvé dans cellules en division active, y compris les jeunes feuilles, apex, l&#39;extrémité des racines et des racines latérales primordia (figure 1). Le modèle non-uniforme sporadique est caractéristique de la phase du cycle cellulaire expression spécifique.

<ol>
	<li>Clough, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Floral+dip:+agrobacterium-mediated+germ+line+transformation.">Floral dip: agrobacterium-mediated germ line transformation.</a> Methods Mol Biol. 286, 91-91 (2005).</li><li>Clough, S. J., Bent, A. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Floral+dip:+a+simplified+method+for+Agrobacterium-mediated+transformation+of+Arabidopsis+thaliana.">Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana.</a> Plant J. 16 (6), 735-735 (1998).</li><li>Wang, C., Liu, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Arabidopsis+ribonucleotide+reductases+are+critical+for+cell+cycle+progression,+DNA+damage+repair,+and+plant+development.">Arabidopsis ribonucleotide reductases are critical for cell cycle progression, DNA damage repair, and plant development.</a> Plant Cell. 18 (2), 350-350 (2006).</li></ol>

L&#39;efficacité de transformation est déterminé par plusieurs facteurs différents, qui sont discutés ci-dessous: <ol><li> La santé des plantes et leur âge sont de première importance. Environ un mois vieilles usines produisant de nombreux bourgeons floraux immatures sont idéales. Découper les pousses primaires pour encourager la formation de pousses secondaires jours 3-4, avant la transformation va augmenter l&#39;efficacité de transformation. Les plants plus âgés donnera lieu à des transformants, mai...

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		<th>Material Name</th>
		<th>Type</th>
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		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
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<tr>
<td>6-benzyladenine</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>852430</td>
<td>&nbsp;</td>
<tr>
<td>Silwet-77</td>
<td>&nbsp;</td>
<td>Crompton Corp.</td>
<td>&nbsp;</td>
<td>Toxic, wear gloves</td>
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<td>Murashige and Skoog Basal medium (MS)</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>M5519</td>
<td>&nbsp;</td>
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<td>Gamborg's vitamin solution 1000X</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>G1019</td>
<td>&nbsp;</td>
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<td>X-Gluc (5-bromo-4-chloro-3-indolyl &beta;-D-glucuronide cyclohexamine salt</td>
<td>&nbsp;</td>
<td>Biosynth</td>
<td>B-7300</td>
<td>Toxic, light sensitive keep in the dark</td>
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<td>Micropore 3M Tape</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>19027761</td>
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floral-dip transformation of arabidopsis thaliana to examine ptso2::&beta;-glucuronidase reporter gene expression

La capacité à introduire des gènes étrangers dans un organisme est le fondement de la biologie moderne et de la biotechnologie. Dans le modèle de plante à fleurs<em&gt; Arabidopsis thaliana</em&gt;, Le floral dip méthode de transformation<sup&gt; 1-2</sup&gt; A remplacé toutes les méthodes précédentes en raison de sa simplicité, l&#39;efficacité et à faible coût. Plus précisément, les pousses de floraison de jeunes<em&gt; Arabidopsis</emPlantes&gt; sont trempées dans une solution de<em&gt; Agrobacterium tumefaciens</em&gt; Transportant spécifiques constructions plasmidiques. Après le trempage, les plantes sont retournées à la croissance normale et les graines de rendement, un petit pourcentage de ce qui sont transformées avec le gène étranger et peut être sélectionné pour le milieu contenant des antibiotiques. Cette méthode floraux-pendage grandement facilité<em&gt; Arabidopsis</em&gt; Recherche et a grandement contribué à notre compréhension de la fonction des gènes des plantes. Dans cette étude, nous utilisons le floral dip méthode pour transformer un gène rapporteur,<em&gt; Β-glucuronidase (GUS)</em&gt;, Sous le contrôle de<em&gt; TSO2</em&gt; Promoteur.<em&gt; TSO2</em&gt;, Codant pour la ribonucléotide réductase (RNR) petite sous-unité<sup&gt; 3</sup&gt;, Est un gène du cycle cellulaire réglementé essentielle pour la biosynthèse PMDN dans la phase S du cycle cellulaire. L&#39;examen des<em&gt; GUS</em&gt; Expression transgénique dans les<em&gt; Arabidopsis</em&gt; Semis montre que<em&gt; TSO2</em&gt; Est exprimé dans les tissus en division active. La méthode expérimentale et de matériaux rapportés peuvent être facilement adaptés non seulement pour la recherche mais aussi pour l&#39;éducation à l&#39;école secondaire et du collégial.

Watch this Scientific Journal Video about Floral-dip Transformation of Arabidopsis thaliana to Examine pTSO2::Î²-glucuronidase Reporter Gene Expression at JoVE.com

Floral-dip Transformation of Arabidopsis thaliana to Examine pTSO2::&amp;#946;-glucuronidase Reporter Gene Expression

Cet article illustre l&#39;floral dip méthode de<em&gt; Agrobacterium tumefaciens</em&gt; Transformation médiée par des<em&gt; Arabidopsis thaliana</em&gt;. En introduisant un cycle cellulaire régulé promoteur-rapporteur,<em&gt; PTSO2:: β-glucuronidase (GUS)</em&gt;, Dans<em&gt; Arabidopsis</em&gt;, Nous montre comment on détecte l&#39;expression rapporteur GUS dans des plants transgéniques.

Floral-pendage de la transformation Arabidopsis thaliana Pour examiner PTSO2:: β-glucuronidase Expression du gène rapporteur

The ability to introduce foreign genes into an organism is the foundation for modern biology and biotechnology. In the model flowering plant Arabidopsis ...

floral-dip-transformation-arabidopsis-thaliana-to-examine-ptso2

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Biology

Floral-dip Transformation of Arabidopsis thaliana to Examine pTSO2::&beta;-glucuronidase Reporter Gene Expression

35.8K vues.  University of Maryland College Park. Cet article illustre l'floral dip méthode de Agrobacterium tumefaciens Transformation médiée par des Arabidopsis thaliana. En introduisant un cycle cellulaire régulé promoteur-rapporteur, PTSO2:: β-glucuronidase (GUS), Dans Arabidopsis, Nous montre comment on détecte l'expression rapporteur GUS dans des plants transgéniques.

Vidéo: Floral-dip Transformation of Arabidopsis thaliana to Examine pTSO2::&#946;-glucuronidase Reporter Gene Expression

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 ...

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Biologie

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

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 ...

Investigating Tissue- and Organ-specific Phytochrome Responses using FACS-assisted Cell-type Specific Expression Profiling in Arabidopsis thaliana

Light mediates an array of developmental and adaptive processes throughout the life cycle of a plant. Plants utilize light-absorbing molecules called photoreceptors to sense and adapt to light. The red/far-red light-absorbing phytochrome photoreceptors have been studied extensively. Phytochromes exist as a family of proteins with distinct and overlapping functions in all higher plant systems in which they have been studied1. Phytochrome-mediated light responses, which range from seed germination through flowering and senescence, are often localized to specific plant tissues or organs2. Despite the discovery and elucidation of individual and redundant phytochrome functions through mutational analyses, conclusive reports on distinct sites of photoperception and the molecular mechanisms of localized pools of phytochromes that mediate spatial-specific phytochrome responses are limited. We designed experiments based on the hypotheses that specific sites of phytochrome photoperception regulate tissue- and organ-specific aspects of photomorphogenesis, and that localized phytochrome pools engage distinct subsets of downstream target genes in cell-to-cell signaling. We developed a biochemical approach to selectively reduce functional phytochromes in an organ- or tissue-specific manner within transgenic plants. Our studies are based on a bipartite enhancer-trap approach that results in transactivation of the expression of a gene under control of the Upstream Activation Sequence (UAS) element by the transcriptional activator GAL43. The biliverdin reductase (BVR) gene under the control of the UAS is silently maintained in the absence of GAL4 transactivation in the UAS-BVR parent4. Genetic crosses between a UAS-BVR transgenic line and a GAL4-GFP enhancer trap line result in specific expression of the BVR gene in cells marked by GFP expression4. BVR accumulation in Arabidopsis plants results in phytochrome chromophore deficiency in planta5-7. Thus, transgenic plants that we have produced exhibit GAL4-dependent activation of the BVR gene, resulting in the biochemical inactivation of phytochrome, as well as GAL4-dependent GFP expression. Photobiological and molecular genetic analyses of BVR transgenic lines are yielding insight into tissue- and organ-specific phytochrome-mediated responses that have been associated with corresponding sites of photoperception4, 7, 8. Fluorescence Activated Cell Sorting (FACS) of GFP-positive, enhancer-trap-induced BVR-expressing plant protoplasts coupled with cell-type-specific gene expression profiling through microarray analysis is being used to identify putative downstream target genes involved in mediating spatial-specific phytochrome responses. This research is expanding our understanding of sites of light perception, the mechanisms through which various tissues or organs cooperate in light-regulated plant growth and development, and advancing the molecular dissection of complex phytochrome-mediated cell-to-cell signaling cascades.

Investigating Tissue- and Organ-specific Phytochrome Responses using FACS-assisted Cell-type Specific Expression Profiling in Arabidopsis thaliana

The molecular basis of spatial-specific phytochrome responses is being investigated using transgenic plants that exhibit tissue- and organ-specific phytochrome deficiencies. The isolation of specific cells exhibiting induced phytochrome chromophore depletion by Fluorescence-Activated Cell Sorting followed by microarray analyses is being utilized to identify genes involved in spatial-specific phytochrome responses.

Étude des tissus et des organes spécifiques Réponses phytochrome utilisant FACS assistée type cellulaire profilage expression spécifique dans Arabidopsis thaliana</em

Light mediates an array of developmental and adaptive processes throughout the life cycle of a plant. Plants utilize light-absorbing molecules called ...

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 ...

Histochemical Staining of Arabidopsis thaliana Secondary Cell Wall Elements

Arabidopsis thaliana is a model organism commonly used to understand and manipulate various cellular processes in plants, and it has been used extensively in the study of secondary cell wall formation. Secondary cell wall deposition occurs after the primary cell wall is laid down, a process carried out exclusively by specialized cells such as those forming vessel and fiber tissues. Most secondary cell walls are composed of cellulose (40&#8211;50%), hemicellulose (25&#8211;30%), and lignin (20&#8211;30%). Several mutations affecting secondary cell wall biosynthesis have been isolated, and the corresponding mutants may or may not exhibit obvious biochemical composition changes or visual phenotypes since these mutations could be masked by compensatory responses. Staining procedures have historically been used to show differences on a cellular basis. These methods are exclusively visual means of analysis; nevertheless their role in rapid and critical analysis is of great importance. Congo red and calcofluor white are stains used to detect polysaccharides, whereas M&#228;ule and phloroglucinol are commonly used to determine differences in lignin, and toluidine blue O is used to differentially stain polysaccharides and lignin. The seemingly simple techniques of sectioning, staining, and imaging can be a challenge for beginners. Starting with sample preparation using the A. thaliana model, this study details the protocols of a variety of staining methodologies that can be easily implemented for observation of cell and tissue organization in secondary cell walls of plants.

Histochemical Staining of Arabidopsis thaliana Secondary Cell Wall Elements

Plant cell wall composition varies between tissue types and can include lignin, cellulose, hemicelluloses, and pectin. Various staining techniques have been developed to visualize differences at the cell-type level. This paper is a compilation of commonly used cell wall staining techniques.

La coloration histochimique de l&#39;<em&gt; Arabidopsis thaliana</em&gt; Cellule secondaire Elements mur

Arabidopsis thaliana is a model organism commonly used to understand and manipulate various cellular processes in plants, and it has been used extensively ...

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 ...

Floral-Dip Transformation of Flax (Linum usitatissimum) to Generate Transgenic Progenies with a High Transformation Rate

Agrobacterium-mediated plant transformation via floral-dip is a widely used technique in the field of plant transformation and has been reported to be successful for many plant species. However, flax (Linum usitatissimum) transformation by floral-dip has not been reported. The goal of this protocol is to establish that Agrobacterium and the floral-dip method can be used to generate transgenic flax. We show that this technique is simple, inexpensive, efficient, and more importantly, gives a higher transformation rate than the current available methods of flax transformation.
In summary, inflorescences of flax were dipped in a solution of Agrobacterium carrying a binary vector plasmid (T-DNA fragment plus the Linum Insertion Sequence, LIS-1) for 1 - 2 min. The plants were laid flat on their side for 24 hr. Then, plants were maintained under normal growth conditions until the next treatment. The process of dipping was repeated 2 - 3 times, with approximately 10 - 14 day intervals between dipping. The T1 seeds were collected and germinated on soil. After approximately two weeks, treated progenies were tested by direct PCR; 2 - 3 leaves were used per plant plus the appropriate T-DNA primers. Positive transformants were selected and grown to maturity. The transformation rate was unexpectedly high, with 50 - 60% of the seeds from treated plants being positive transformants. This is a higher transformation rate than those reported for Arabidopsis thaliana and other plant species, using floral-dip transformation. It is also the highest, which has been reported so far, for flax transformation using other methods for transformation.

Floral-Dip Transformation of Flax Linum usitatissimum to Generate Transgenic Progenies with a High Transformation Rate

Here, we present a protocol to transform flax using Agrobacterium-mediated plant transformation via floral-dip. This protocol is simple to perform and inexpensive, yet yields a higher transformation rate than the current available methods for flax transformation.

Floral-Dip Transformation de lin (<em&gt; Linum usitatissimum</em&gt;) Pour générer transgéniques descendances avec un taux de transformation élevé

Agrobacterium-mediated plant transformation via floral-dip is a widely used technique in the field of plant transformation and has been reported to be ...

Visualizing Stromule Frequency with Fluorescence Microscopy

Stromules, or "stroma-filled tubules", are narrow, tubular extensions from the surface of the chloroplast that are universally observed in plant cells but whose functions remain mysterious. Alongside growing attention on the role of chloroplasts in coordinating plant responses to stress, interest in stromules and their relationship to chloroplast signaling dynamics has increased in recent years, aided by advances in fluorescence microscopy and protein fluorophores that allow for rapid, accurate visualization of stromule dynamics. Here, we provide detailed protocols to assay stromule frequency in the epidermal chloroplasts of Nicotiana benthamiana, an excellent model system for investigating chloroplast stromule biology. We also provide methods for visualizing chloroplast stromules in vitro by extracting chloroplasts from leaves. Finally, we outline sampling strategies and statistical approaches to analyze differences in stromule frequencies in response to stimuli, such as environmental stress, chemical treatments, or gene silencing. Researchers can use these protocols as a starting point to develop new methods for innovative experiments to explore how and why chloroplasts make stromules.

Protocols to investigate the dynamics of chloroplast stromules, the stroma-filled tubules that extend from the surface of chloroplasts, are described.

Visualisation Fréquence Stromule avec Fluorescence Microscopy

Stromules, or "stroma-filled tubules", are narrow, tubular extensions from the surface of the chloroplast that are universally observed in plant cells but ...

Etude spatio-temporelle de la dynamique d’ERL1 via l’imagerie confocale

Image-Based Methods to Study Membrane Trafficking Events in Stomatal Lineage Cells

In eukaryotic cells, membrane components, including proteins and lipids, are spatiotemporally transported to their destination within the endomembrane system. This includes the secretory transport of newly synthesized proteins to the cell surface or the outside of the cell, the endocytic transport of extracellular cargoes or plasma membrane components into the cell, and the recycling or shuttling transport of cargoes between the subcellular organelles, etc. Membrane trafficking events are crucial to the development, growth, and environmental adaptation of all eukaryotic cells and, thus, are under stringent regulation. Cell-surface receptor kinases, which perceive ligand signals from the extracellular space, undergo both secretory and endocytic transport. Commonly used approaches to study the membrane trafficking events using a plasma membrane-localized leucine-rich-repeat receptor kinase, ERL1, are described here. The approaches include plant material preparation, pharmacological treatment, and confocal imaging setup. To monitor the spatiotemporal regulation of ERL1, this study describes the co-localization analysis between ERL1 and a multi-vesicular body marker protein, RFP-Ara7, the time series analysis of these two proteins, and the z-stack analysis of ERL1-YFP treated with the membrane trafficking inhibitors brefeldin A and wortmannin.

Pleins feux sur l’auteur : Méthodes basées sur l’image pour étudier les événements de trafic membranaire dans les cellules de la lignée stomatique  

Several commonly used methods are introduced here to study the membrane trafficking events of a plasma membrane receptor kinase. This manuscript describes detailed protocols including the plant material preparation, pharmacological treatment, and confocal imaging setup.

Méthodes basées sur l’image pour étudier les événements de trafic membranaire dans les cellules de la lignée stomatique

In eukaryotic cells, membrane components, including proteins and lipids, are spatiotemporally transported to their destination within the endomembrane ...