Wir danken Chunxin Wang für den Bau pTSO2:: GUS und für Fotos in Abbildung 1, Detlef Weigel für die gemeinsame Nutzung der GUS-narrensicher Protokoll, Paja Sijacic und Courtney Hollender für hilfreiche Kommentare. Research in Z. L s Labor ist von der US National Science Foundation (IOB0616096 und MCB0744752) unterstützt. ZL wird teilweise durch die University of Maryland Agricultural Experiment Station unterstützt.

I. Floral-dip Transformation von Arabidopsis thaliana <ol><li> Etwa 1 Monat vor der Transformation, säen Arabidopsis Samen auf 4 bis 8 Töpfe (5 Zoll Vierkant-Töpfe) mit ca. 10-15 Pflanzen pro Topf. Für Anlagen mit normaler Fruchtbarkeit, werden 4 Töpfe mit Pflanzen pro Plasmid-Konstrukt ausreichend sein. Wenn man mutierte Arabidopsis mit reduzierter Fruchtbarkeit zu verwandeln, erhöhen Sie die Anzahl der Töpfe entsprechend. </li><li> Die Pflanzen werden unter Standard-Bedingungen (16 Stunden Licht und 8 h Dunkelheit bei 22 ° C) gewachsen. Wenn Pflanzen unter kürzeren Tag oder niedrigere Temperatur gezüchtet werden, werden sie länger dauern, zu blühen und mehr bereit zu sein für die Transformation. Gute Anlage Pflege garantieren gesunde Pflanzen, die wesentlich für eine erfolgreiche Transformation sind. </li><li> Wenn die Pflanzen nur verschraubt und angefangen zu blühen. Sie sind nun bereit für die Transformation. Zur Erhöhung der Transformationseffizienz, 3-4 Tage vor der Transformation, schneiden Sie die Hauptinfloreszenz schießt, sobald sie verschraubt mehrere sekundäre Sproßbildung fördern. Wasser die Pflanzen am Tag vor der Transformation. Dadurch wird sichergestellt, dass der Boden nicht tanken zu viel Infiltration Medien während floral-Tauchen. </li><li> Zwei Tage vor der Transformation, impfen 40ml LB mit geeigneten Antibiotika (im Falle von pTSO2:: GUS, 50 ug / ml Kanamycin) mit Agrobacterium tumefaciens Stamm GV3101 Durchführung der pTSO2:: GUS-Konstrukt. Die pTSO2:: GUS wird in der pBIN20 Vektor, NPTII Gen, das Resistenz gegen Kanamycin 3 besitzt geklont. Wachsen Sie über Nacht in einem Schüttelinkubator bei 28-30 ° C. </li><li> Am folgenden Tag, Transfer 8ml der Übernacht-Kultur in 400 ml LB mit 50 ug / ml Kanamycin. Wachsen Sie über Nacht in einem Schüttelinkubator bei 28-30 ° C. </li><li> Am Tag der Transformation, wenn die OD 600 der Agrobacterium Kultur rund 0,8 (OD 600 zwischen 0,5 zu 1 sind zulässig) erreicht, Spin-down der Agrobacterium-Nacht-Kultur in 8 Minuten bei 5000rpm. </li><li> Resuspendieren Agrobacterium Pellet in 1 L (Liter) der Infiltration Medien (10 mM MgCl 2, 5% Saccharose, 0.44 mm 6-benzyladenine (BA), 0,3% Silwet L-77, 1x Gamborg s Vitamin-Lösung und autoklaviert Wasser). Das Medium muss nicht sterilisiert werden, sondern muss frisch zubereitet werden. Gießen Sie die 1 L Agrobacterium-haltigen Infiltration Medium in eine Schale (z. B. 8 &quot;x15&quot; x2 &quot;Pyrex Glasschale). </li><li> Kehren Sie die Pot (Pflanzen fällt nicht aus) und tauchen Sie alle oberirdischen Teile der Pflanzen in die Infiltration Lösung für 5 Minuten einen Topf zu halten in einer Zeit. Während alle oberirdischen Teile der Pflanze in die Infiltration Medium eingetaucht sind, verhindern, dass Boden aufzusaugen der Infiltration Medien, um die Chancen von Pilzwachstum auf den Boden zu reduzieren. </li><li> Nach dem Eintauchen Ort alle getaucht Töpfe auf ihrer Seite auf mehrere Lagen Papier Handtuch in ein Fach ein. Die Papiertücher tanken Zugang Menge Infiltration Medien. Decken Sie die Schale mit Frischhaltefolie zu hohe Luftfeuchtigkeit zu gewährleisten. Setzen Sie das Fach in das Pflanzenwachstum Kammer. </li><li> Am folgenden Tag, entfernen Sie die Plastikfolie und Papierhandtücher, Ort und Töpfe aufrecht. Nicht Wasser dieser Anlagen für vier vor fünf weitere Tage. Danach halten die Pflanzen in der Regel, bis sie Samen gesetzt und sammeln Samen in loser Schüttung. </li><li> Platten gießen (150x15 Petrischale) mit MS-Medium mit 50 ug / ml Kanamycin (Gewicht 2,2 g Murashige und Skoog basalen Salz ohne Vitamin auflösen in 500 ml Wasser, pH auf 5,8 mit 1 M NaOH, fügen Sie 4 Gramm Agar, Autoklaven, kühl und add Kanamycin bis 50 ug / ml Endkonzentration). Die Platten werden in 4 ° C gehalten, bis sie benötigt. </li><li> So wählen Sie für die Antibiotika-resistenten transgenen Pflanzen, braucht man zu 20.000 Samen mindestens Bildschirm. 0,1 Gramm ist ungefähr 5000 Samen. Gewicht und schätzen die Menge an Samen. </li><li> Sterilisieren Samen durch waschen Sie sie in 15 ml Falcon-Röhrchen mit 70% Ethanol für 30 Sekunden. Wash Samen mit sterilem Wasser wieder. Weichen Sie die Samen für 30 Sekunden in eine Lösung, die 1:10 Verdünnung der im Laden gekauften 5% Bleichmittel (Clorox). Spülen Sie Samen mit sterilem Wasser drei Minuten vor sechs mal. In sterile Haube, ausgebreitet Pipette ca. 5000 Samen auf jedes MS (Kanamycin) Platte, die Samen gleichmäßig auf der Platte, dichten die Platten mit Micropore 3M Klebeband, um eine Kontamination zu vermeiden. </li><li> Die Inkubation bei 4 ° C im Dunkeln für 3-4 Tage, dann überweisen Sie den Teller mit einer Kammer (könnte das gleiche Pflanzenwachstum Kammer werden). Nach etwa 14 Tagen, bleiben transgenen Keimlinge grün, aber der nicht-transgenen diejenigen sind blaß und Sterben. Übertragen Sie die transgenen Keimlinge in den Boden durch Verlangsamung Ziehen der Wurzel aus dem mittleren und legte sie im Boden. </li></ol> II. Untersuchen pTSO2:: GUS-Expressionsmuster <ol><li> Ernte pTSO2:: GUS transgenen Keimlinge und legen Sie sie in kaltem 90% Aceton in einem Glasszintillationsröhrchen oder Mikrozentrifugenröhrchen, die auf Eis gehalten. Polystyrol-Mikrotiterplatten (nicht Polypropylen) kann auch verwendet werden, wenn man analysiert eine große Anzahl von Proben sein. </li><li> Nachdem alle Proben entnommen werden, legen Sie die Fläschchen bei Raumtemperatur für 20 Minuten. Während dieser Zeit bilden frische Färbepuffer ohne x-Gluc (0,2% Triton x-100, 50 mM NaHPO 4 Buffer pH 7,2, 2 mM Kaliumhexacyanoferrat, 2mM Kaliumferricyanid) und auf Eis stellen. </li><li> Entfernen Aceton aus den Proben und fügen Sie die Färbepuffer. </li><li> Make up x-Gluc-Färbelösung (0,2% Triton x-100, 50 mM NaHPO 4 Buffer pH 7,2, 2 mM Kaliumhexacyanoferrat, 2mM Kaliumferricyanid, 2 mM X-Gluc). Entfernen Sie die Färbepuffer von Proben und fügen Sie die Färbung Puffer mit x-Gluc zu den Proben. </li><li> Infiltrate der Proben unter Vakuum für 15 bis 20 Minuten. Lassen Sie die Vakuum langsam und sicher, dass alle Proben unter der Oberfläche der Färbelösung sinken. Falls erforderlich, wiederholen Infiltration, bis alle Proben sinken, nachdem das Vakuum freigesetzt. </li><li> Inkubieren Proben bei 37 ° C über Nacht in die Färbepuffer mit x-Gluc. </li><li> Nehmen Sie Proben von Inkubator und entfernen Färbepuffer. Waschen Sie die Proben in aufeinanderfolgenden Ethanol-Reihe (20%, 35% und 50% Ethanol) bei Raumtemperatur für jeweils 30 Minuten ändern. </li><li> Fix das Gewebe in FAA Fixativ (50% Ethanol, 5% Formaldehyd, 10% Essigsäure, Rest Wasser) für 30 Minuten bei Raumtemperatur. Nehmen Sie FAA und fügen 70% Ethanol. An dieser Stelle im Gewebe sichtbar gemacht werden und fotografiert unter einem Binokular mit einer digitalen Kamera ausgestattet. Eine dunkelblaue Farbe zeigt an, wo TSO2 exprimiert wird (Abbildung 1). Alternativ kann das Gewebe bei 4 ° C für die spätere Ansicht gespeichert werden. <img src="/files/ftp_upload/1952/1952fig1.jpg" alt="Abbildung 1" /> Abbildung 1 pTSO2.:: GUS-Expression in transgenen Keimlingen (A), seitliche Wurzeln (B), und Wurzelspitze (C). Beachten Sie, dass TSO2 Promoter hochaktive in jungen und Dividieren Gewebe ist. Die sporadischen Expressionsmuster in (C) gezeigt, ist typisch von Genen in bestimmten Zellzyklusphasen ausgedrückt. </li></ol> Repräsentative Ergebnisse Wenn es richtig gemacht, sollte Transformationseffizienz ca. 0,1-0,2% sein. Mit anderen Worten, sollte man 5-10 transgenen Keimlinge durch Screening alle 5000 Samen erhalten. Im Durchschnitt können etwa 100 transgenen Linien durch die Umwandlung 4 Töpfe von Wildtyp-Pflanzen gewonnen werden. Für transgenen Keimlinge mit den pTSO2:: GUS-Konstrukt 3, dunkelblaue Farbe reflektiert GUS-Aktivität ist in sich aktiv teilenden Zellen, einschließlich jungen Blätter, Sprosszellen, Wurzelspitze und lateralen Wurzel Primordien (Abbildung 1). Die ungleichmäßige sporadischen Muster ist charakteristisch für Zellzyklusphase-spezifische Expression.

<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>

Die Effizienz der Transformation wird durch viele verschiedene Faktoren, die im Folgenden diskutiert bestimmt: <ol><li> Die Gesundheit der Pflanzen und deren Alter von primärer Bedeutung sind. Etwa einen Monat alten Anlagen zur Produktion von zahlreichen unreifen Blütenknospen sind ideal. Trimmen Sie primäre Triebe zu fördern sekundären Sproßbildung 3-4 Tage vor der Transformation erhöht Transformationseffizienz. Ältere Pflanzen werden steigen, um Transformanten zu geben, sondern zu einem niedrigeren Zinssat...

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		<th>Catalogue Number</th>
		<th>Comment</th>
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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>
<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>
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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

Die Fähigkeit, fremde Gene in einen Organismus einbringen ist das Fundament für die moderne Biologie und Biotechnologie. In das Modell blühende Pflanze<em&gt; Arabidopsis thaliana</em&gt;, Die floral-dip Transformationsmethode<sup&gt; 1-2</sup&gt; Hat alle bisherigen Methoden wegen seiner Einfachheit, Effizienz und geringen Kosten ersetzt. Genauer gesagt, schießt der junge Blüte<em&gt; Arabidopsis</em&gt; Pflanzen werden in einer Lösung aus getaucht<em&gt; Agrobacterium tumefaciens</em&gt; Durchführung spezifischer Plasmid-Konstrukte. Nach dem Eintauchen werden die Pflanzen auf ein normales Wachstum und besserem Saatgut, ein kleiner Prozentsatz der die mit dem fremden Gen transformiert und können bei Medium mit Antibiotika ausgewählt werden zurückgegeben. Das blumig-dip-Methode wesentlich erleichtert<em&gt; Arabidopsis</em&gt; Forschung und trug wesentlich zu unserem Verständnis der pflanzlichen Gen-Funktion. In dieser Studie verwenden wir die floral-Tauchverfahren mit einem Reportergen zu verwandeln,<em&gt; Β-Glucuronidase (GUS)</em&gt;, Unter der Kontrolle von<em&gt; TSO2</em&gt; Promotor.<em&gt; TSO2</em&gt;, Kodierend für die Ribonucleotid-Reductase (RNR) kleine Untereinheit<sup&gt; 3</sup&gt;, Ist ein Zellzyklus reguliert essentielles Gen für die Biosynthese dNDP in der S-Phase des Zellzyklus. Untersuchung der<em&gt; GUS</em&gt; Expression in transgenen<em&gt; Arabidopsis</em&gt; Keimlinge zeigt, dass<em&gt; TSO2</em&gt; Ist in sich aktiv teilenden Geweben exprimiert. Die berichteten experimentellen Methode und Materialien können problemlos nicht nur für die Forschung, sondern auch für die Bildung werden an der High School und College-Niveau angepasst.

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

Dieser Artikel veranschaulicht die floral-dip-Methode der<em&gt; Agrobacterium tumefaciens</em&gt;-Vermittelte Transformation von<em&gt; Arabidopsis thaliana</em&gt;. Durch die Einführung eines Zellzyklus-regulierten Promotor-Reporter<em&gt; PTSO2: β-Glucuronidase (GUS)</em&gt;, In<em&gt; Arabidopsis</em&gt;, Zeigt uns, wie man GUS-Reporter Expression in transgenen Keimlinge erkennt.

Floral-dip Transformation von Arabidopsis thaliana Zum Untersuchen PTSO2: β-Glucuronidase Reporter Gene Expression

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

Research

JoVE Journal

Biology

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

36.0K Aufrufe.  University of Maryland College Park. Dieser Artikel veranschaulicht die floral-dip-Methode der Agrobacterium tumefaciens-Vermittelte Transformation von Arabidopsis thaliana. Durch die Einführung eines Zellzyklus-regulierten Promotor-Reporter PTSO2: β-Glucuronidase (GUS), In Arabidopsis, Zeigt uns, wie man GUS-Reporter Expression in transgenen Keimlinge erkennt.

Serie: 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.

Die Herstellung von primären hämatopoetischen Zellkulturen aus Knochenmark der Maus für die Elektroporation

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

Forschung

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.

Mit dem Gene Pulser MXcell Elektroporation System zur Primary Cells mit hohem Wirkungsgrad zu transfizieren

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.

Verwendung eines automatisierten Cell Counter zu Genexpressionsstudien Simplify: siRNA Knockdown von IL-4 abhängige Genexpression in Namalwalzellen

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.

Untersuchung Tissue-und Organ-spezifische Phytochrom Antworten mit FACS-gestützte zelltypspezifisch Expression Profiling 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.

In vitro Rekonstitution der 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 ...

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.

Histochemische Färbung von<em&gt; Arabidopsis thaliana</em&gt; Sekundärzellwand-Elemente

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 der globalen RNA-Synthese auf der Einzelzellebene folgende 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 von Flachs (<em&gt; Linum usitatissimum</em&gt;), Um Transgene Progenies mit hoher Umwandlungsrate generieren

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.

Visualizing Stromulus Frequenz mit Fluoreszenzmikroskopie

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

Raum-zeitliche Untersuchung der ERL1-Dynamik mittels konfokaler Bildgebung

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.

Autor im Rampenlicht: Bildbasierte Methoden zur Untersuchung von Membrantransportereignissen in Zellen der Stomata-Linie  

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.

Bildbasierte Methoden zur Untersuchung von Membrantransportereignissen in stomatalen Zellen

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