Ringraziamo Chunxin Wang per la costruzione di pTSO2:: GUS e per le foto in figura 1, Detlef Weigel per la condivisione del GUS-infallibile protocollo, Paja Sijacic, e Courtney Hollender per gli utili commenti. La ricerca in laboratorio, Z. L s è supportato dal US National Science Foundation (IOB0616096 e MCB0744752). ZL è parzialmente supportata dalla University of Maryland Stazione esperimento agricola.

I. floreale-dip trasformazione di Arabidopsis thaliana <ol><li> Circa 1 mese prima trasformazione, seminare i semi di Arabidopsis sul 4-8 pentole (5 vasi pollice quadrato), con circa 10-15 piante per vaso. Per gli impianti con la fertilità normale, 4 vasi di piante per costruire plasmide sarà sufficiente. Se uno ha a trasformare Arabidopsis mutante con riduzione della fertilità, aumentare il numero di vasi di conseguenza. </li><li> Le piante sono coltivate in condizioni standard (16 ore di luce e 8 ore al buio a 22 ° C). Se le piante sono coltivate in brevi giorno o abbassare la temperatura, che richiederà più tempo a fiorire e più tempo per essere pronti per la trasformazione. La cura delle piante buona volontà assicurare piante sane, che sono essenziali per la trasformazione di successo. </li><li> Quando le piante hanno appena cominciato a imbullonati e fiori. Essi sono ora pronti per la trasformazione. Per aumentare l&#39;efficienza della trasformazione, 3-4 giorni prima della trasformazione, tagliare i tralci principali infiorescenza non appena sono imbullonati per incoraggiare la formazione di scattare più secondario. Annaffiare le piante il giorno prima trasformazione. Questo assicura che il terreno non assorbire i media infiltrazione troppo durante floreali-immersione. </li><li> Due giorni prima della trasformazione, inoculare LB 40ml contenenti antibiotici appropriati (nel caso di pTSO2:: GUS, 50 mg / ml kanamicina) con Agrobacterium tumefaciens ceppo GV3101 porta la pTSO2:: GUS costruire. La pTSO2:: GUS è clonato nel vettore pBIN20 che possiede gene nptII che conferisce resistenza alla kanamicina 3. Crescere durante la notte in una agitazione incubatore a 28-30 ° C. </li><li> Il giorno seguente, 8ml trasferimento della cultura durante la notte in 400 ml di LB contenente 50 mg / ml kanamicina. Crescere durante la notte in una agitazione incubatore a 28-30 ° C. </li><li> Il giorno della trasformazione, quando il diametro esterno 600 della cultura Agrobacterium raggiunge circa 0,8 (OD 600 tra 0,5 a 1 sono accettabili), spin down la cultura Agrobacterium pernottamento per 8 minuti a 5000rpm. </li><li> Risospendere il pellet Agrobacterium in 1 L (litri) di media infiltrazione (10mM MgCl 2, 5% di saccarosio, 0.44mM 6-benziladenina (BA), 0,3% Silwet L-77, soluzione di vitamina 1x Gamborg s acqua e autoclave). Il mezzo non ha bisogno di essere sterilizzati in autoclave, ma deve essere fatta fresca. Versare la L 1 Agrobacterium contenente medio infiltrazione in un piatto (come 8 &quot;x15&quot; x2 &quot;piatto di vetro Pyrex). </li><li> Invertire il vaso (piante non cadere) e tuffo tutte le parti aeree delle piante nella soluzione infiltrazione, tenere premuto per 5 minuti un piatto alla volta. Mentre tutte le parti aeree della pianta sono immersi nel mezzo infiltrazione, evitare di lasciare assorbire il terreno dei media infiltrazione per ridurre le possibilità di proliferazione di funghi sul terreno. </li><li> Dopo l&#39;immersione, luogo immerso tutte le pentole al loro fianco in diversi strati di carta assorbente in un vassoio. I tovaglioli di carta assorbire quantità accesso dei mezzi di infiltrazione. Coprire il vassoio con pellicola trasparente per assicurare elevata umidità. Collocare il vassoio in camera di crescita delle piante. </li><li> Il giorno seguente, rimuovere l&#39;involucro di plastica e tovaglioli di carta, pentole e posto in posizione verticale. Non acqua queste piante per quattro o cinque più giorni. In seguito, mantenere le piante in genere fino a quando non impostare semi e raccogliere i semi alla rinfusa. </li><li> Versare nelle piastre (150x15mm capsula di Petri), con media MS contenente 50 mg / ml kanamicina (peso 2.2g Murashige e Skoog sale basale senza vitamina, sciogliere in 500 ml di acqua, il pH a 5,8 con NaOH 1M, aggiungere 4 grammi di agar, autoclave, fresco e aggiungere kanamicina a 50 mg / ml concentrazione finale). Le piastre sono tenuti in 4 ° C fino al momento dell&#39;uso. </li><li> Per selezionare le piante transgeniche resistenti agli antibiotici, bisogna schermo 20.000 semi al minimo. 0,1 grammi è di circa 5000 semi. Peso e stimare la quantità di semi. </li><li> Sterilizzare i semi di lavarli in 15 ml di tubo Falcon con il 70% di etanolo per 30 secondi. Lavare i semi con acqua sterile una volta. Mettere a bagno i semi per 30 secondi in una soluzione contenente diluizione 1:10 del negozio-acquistato candeggina al 5% (candeggina). Sciacquare i semi con acqua sterile tre a sei volte. Nel cappuccio sterile, pipetta circa 5000 semi su ogni (kanamicina) piastra SM, diffondere i semi in modo uniforme sul piatto, sigillare le piastre con nastro adesivo 3M Micropore per evitare contaminazioni. </li><li> Incubare le piastre a 4 ° C al buio per 3-4 giorni, poi trasferire le piastre di una camera (potrebbe essere la stessa camera di crescita delle piante). Dopo circa 14 giorni, piantine transgeniche soggiorno verde ma la non-transgenico quelli si stanno rivolgendo pallido e morente. Trasferire le piantine transgeniche al suolo rallentando tirando le radici fuori dal mezzo e li pose nel suolo. </li></ol> II. Esaminando pTSO2:: pattern di espressione GUS <ol><li> Vendemmia pTSO2:: GUS piantine transgeniche e metterli in acetone freddo il 90% in un flacone di vetro o tubi di scintillazione microcentrifugache vengono tenuti in ghiaccio. Micropiastre di polistirene (non polipropilene) può essere utilizzato anche se si analizza un numero elevato di campioni. </li><li> Dopo che tutti i campioni vengono raccolti, posto le fiale a temperatura ambiente per 20 minuti. Durante questo periodo compongono tampone fresca colorazione senza x-Gluc (0,2% Triton X-100, 50mm NaHPO 4 Buffer pH7.2, 2mM ferrocianuro di potassio, 2mM ferricianuro di potassio) e posto sul ghiaccio. </li><li> Rimuovere acetone dai campioni e aggiungere la soluzione tampone colorazione. </li><li> Make up x-Gluc soluzione colorante (0,2% Triton X-100, 50mm NaHPO 4 Buffer pH7.2, Potassio Ferrocianuro 2mM, 2mM ferricianuro di potassio, 2mm x-Gluc). Rimuovere il tampone colorazione da campioni e aggiungere la soluzione tampone contenente colorazione x-Gluc ai campioni. </li><li> Infiltrati i campioni sotto vuoto per 15 a 20 minuti. Rilasciare lentamente il vuoto e verificare che tutti i campioni affondare sotto la superficie della soluzione colorante. Se necessario, ripetere l&#39;infiltrazione fino a quando tutti affondare campioni dopo il vuoto è stato rilasciato. </li><li> Campioni incubare a 37 ° C per una notte nel buffer di colorazione con x-Gluc. </li><li> Rimuovere i campioni da incubatore e rimuovere buffer di colorazione. Lavare i campioni in serie successive di etanolo (20%, 35% e 50% di etanolo) a temperatura ambiente per 30 minuti ogni modifica. </li><li> Fissare i tessuti in FAA fissativo (50% etanolo, 5% di formaldeide, 10% di acido acetico, acqua riposo) per 30 minuti a temperatura ambiente. Rimuovere FAA e aggiungere etanolo al 70%. A questo punto i tessuti possono essere visualizzati e fotografato con un microscopio da dissezione dotato di una fotocamera digitale. Un colore blu scuro indica dove TSO2 è espressa (Figura 1). In alternativa, i tessuti possono essere conservati a 4 ° C per una visione successiva. <img src="/files/ftp_upload/1952/1952fig1.jpg" alt="Figura 1" /> Figura 1 pTSO2:.: Espressione di GUS in piantine transgeniche (A), radici laterali (B), e la punta della radice (C). Si noti che TSO2 promotore è molto attivo nei tessuti giovani e dividendo. Il pattern di espressione sporadici in (C) è tipica di geni espressi in specifiche fasi del ciclo cellulare. </li></ol> Rappresentante Risultati Se fatto correttamente, l&#39;efficienza della trasformazione dovrebbe essere di circa 0,1-0,2%. In un&#39;altra parola, si dovrebbe avere 50-10 piantine transgeniche attraverso lo screening ogni 5000 semi. In media, circa 100 linee transgeniche può essere ottenuto trasformando 4 vasi di piante wild-type. Per le piantine transgeniche contenenti il pTSO2:: GUS costruire 3, colore blu scuro che riflette l&#39;attività GUS si trova nelle cellule in divisione attiva tra giovani foglie, all&#39;apice sparare, punta della radice, e laterale radice primordi (Figura 1). La non uniforme modello sporadiche è caratteristico del ciclo cellulare fase-specifici espressione.

<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>

Nessun conflitto di interessi dichiarati.

L&#39;efficienza di trasformazione è determinato da molti fattori diversi, che sono discussi di seguito: <ol><li> La salute delle piante e la loro età sono di primaria importanza. Circa un mese impianti di produzione di numerose gemme floreali immaturi sono l&#39;ideale. Taglio fuori spara primario di incoraggiare secondaria giornate di formazione sparare 3-4 prima della trasformazione aumenterà l&#39;efficienza della trasformazione. Vecchi impianti darà origine a trasformanti ma ad un tasso inferiore. </li><li>...

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		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<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>
</tr>
<tr>
<td>Murashige and Skoog Basal medium (MS)</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>M5519</td>
<td>&nbsp;</td>
<tr>
<td>Gamborg's vitamin solution 1000X</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>G1019</td>
<td>&nbsp;</td>
<tr>
<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>
</tr>
<tr>
<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 possibilità di introdurre geni estranei in un organismo è il fondamento della biologia moderna e le biotecnologie. Nella pianta fiorita modello<em&gt; Arabidopsis thaliana</em&gt;, I fiori-dip trasformazione del metodo<sup&gt; 1-2</sup&gt; Ha sostituito tutti i metodi precedenti per la sua semplicità, efficienza e basso costo. In particolare, i germogli di fioritura giovani<em&gt; Arabidopsis</emPiante&gt; sono immersi in una soluzione di<em&gt; Agrobacterium tumefaciens</em&gt; Trasporto specifico costruisce plasmide. Dopo l&#39;immersione, le piante vengono restituiti alla crescita normale e semi di resa, una piccola percentuale dei quali sono trasformate con il gene estraneo e possono essere selezionati per il supporto contenente antibiotici. Questo floreali-dip metodo significativamente facilitato<em&gt; Arabidopsis</emDi ricerca&gt; e ha contribuito notevolmente alla comprensione della funzione dell&#39;impianto gene. In questo studio, si usa il floreali-dip metodo per trasformare un gene reporter,<em&gt; Β-glucuronidasi (GUS)</em&gt;, Sotto il controllo di<em&gt; TSO2</em&gt; Promotore.<em&gt; TSO2</em&gt;, Che codifica per la ribonucleotide reduttasi (RNR) subunità piccola<sup&gt; 3</sup&gt;, È un gene del ciclo cellulare regolata essenziale per la biosintesi dNDP nella fase S del ciclo cellulare. Esame della<em&gt; GUS</emEspressione&gt; in transgenico<em&gt; Arabidopsis</em&gt; Piantine mostra che<em&gt; TSO2</em&gt; È espressa in modo attivo divisione tessuti. Il metodo riportato sperimentale e materiali può essere facilmente adattato non solo per la ricerca ma anche di istruzione alle scuole superiori e livelli di college.

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

Questo articolo illustra la floreali-dip metodo di<em&gt; Agrobacterium tumefaciens</em&gt;-Mediata trasformazione del<em&gt; Arabidopsis thaliana</em&gt;. Con l&#39;introduzione di un ciclo cellulare regolata promotore-reporter,<em&gt; PTSO2:: β-glucuronidasi (GUS)</em&gt;, In<em&gt; Arabidopsis</em&gt;, Ci illustra come si rileva giornalista espressione GUS in piantine transgeniche.

Floreale-dip Trasformazione Arabidopsis thaliana Per esaminare PTSO2:: β-glucuronidasi Reporter espressione genica

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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JoVE Journal

Biology

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

36.0K Views.  University of Maryland College Park. Questo articolo illustra la floreali-dip metodo di Agrobacterium tumefaciens-Mediata trasformazione del Arabidopsis thaliana. Con l'introduzione di un ciclo cellulare regolata promotore-reporter, PTSO2:: β-glucuronidasi (GUS), In Arabidopsis, Ci illustra come si rileva giornalista espressione GUS in piantine transgeniche.

Video: 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 preparazione di colture primarie di cellule emopoietiche dal midollo osseo murino per elettroporazione

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

Ricerca

Biologia

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.

Utilizzando il sistema di elettroporazione Gene Pulser MXcell per trasfettare cellule primarie con alta efficienza

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.

L&#39;utilizzo di un contatore automatico cella per semplificare gli studi sull&#39;espressione genica: Knockdown siRNA di IL-4 genica dipendente nelle celle 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.

Indagare tessuti e risposte fitocromo organo-specifici utilizzando FACS-Cell-assistita di tipo profili specifici di espressione in 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.

Ricostituzione in vitro di Active T. castaneum telomerasi

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.

Colorazione istochimica di<em&gt; Arabidopsis thaliana</em&gt; Cellula secondaria Elementi da parete

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.

Analisi globale di RNA Sintesi a livello di singola cellula seguente ipossia

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.

Trasformazione Floral-Dip di lino (<em&gt; Linum usitatissimum</em&gt;) Per generare transgenici progenie con una trasformazione Tasso alta

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.

Visualizzazione Frequenza Stromule con microscopia a fluorescenza

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

Indagine spaziotemporale della dinamica ERL1 tramite imaging 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.

Autore in primo piano: Metodi basati su immagini per studiare gli eventi di traffico di membrana nelle cellule del lignaggio stomatico  

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.

Metodi basati su immagini per studiare eventi di traffico di membrana in cellule di lignaggio stomatico

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