Die Autoren bedanken sich bei Dr. Chris Taylor (Ohio State University) für die Einführung von hairy root-Technologie in unser Labor und Drs danken. Senthil Subramanian (South Dakota State University), Juan Zhang (Ludong Universität Shandong China) zur Verbesserung des Protokolls. Wir danken auch Dr. Hao Cheng (Nanjing Agriculture University) für hilft bei der Herstellung dieses Video. Diese Arbeit wird teilweise durch Zuschüsse aus DOE (DE-SC0001295), NSF (MCB-0923779) und USDA (2010-65116-20514) zu OY unterstützt

Das folgende Protokoll wurde verwendet, um hairy root Composite-Anlagen erzeugen in einem Modell Leguminosenarten M. truncatula. Ähnliche Protokolle für mindestens acht Pflanzenarten 1-4 angepasst. Wir benutzten M. truncatula hairy root Composite Pflanzen zu studieren Genfunktionen in Wurzel und Knötchen Entwicklung. Das Protokoll wurde in vier Abschnitte unterteilt: 1) Vorbereitung pflanzlichen Materialien, 2) erzeugt hairy root Korbblütler, 3) symbiontischen Rhizobien Infektion und 4) transgenen Wurzel Identifikation. Wir verwendeten den binären Vektor mit dem grün fluoreszierenden Protein (GFP)-Gen als Reporter für das Screening von transgenen Wurzel in Composite-Pflanzen 3. Im Vergleich zu Antibiotika-Selektion ist GFP-basierte Screening schnell, einfach und kostengünstig. In unserem konstruieren, ist die ER-Ausdruck-optimierte GFP-Gen durch die Super-Ubiquitin-Promotor, die starken konstitutiven GFP-Signale in transgenen Wurzeln getrieben hat, was eine einfache Unterscheidung zwischen transgenen und nicht transgenen Wurzeln. 1. Vorbereitung Werk Materials <ol><li> M. truncautla Samen von Pflanzen in einem Gewächshaus (50% relative Luftfeuchtigkeit, 16 / 8 h hell / dunkel, 22/18 ° C Tag / Nacht-Temperatur) geerntet. Reife Samen können bei 4 ° C gelagert werden </li><li> Etwa 100 Samen werden in 10 ml konzentrierter Schwefelsäure (95-98%, Sigma-Aldrich, MO) für 10 min mit periodischen Schütteln scarified. </li><li> Die Samen sind 5-7 mal mit sterilem Wasser unter leichtem Rühren gespült. </li><li> Die Samen werden dann in Wasser bei Raumtemperatur getränkt für 6 Stunden und bei 4 ° C für 36-48 Stunden, um die Keimung zu synchronisieren. </li><li> Ca. 20-30 Samen werden auf feuchtem Filterpapier Papiere in einer Petrischale ausgebreitet. Sie sind bei 22 ° C gehalten, mit 16 / 8 h Licht / Dunkel-Zyklus und 40 μmol.m -2. S -1 Lichtintensität, für 5-6 Tage. </li><li> Gekeimten Sämlinge werden in den Boden überführt und im Gewächshaus für einen Monat. </li></ol> 2. Generieren von Hairy Root-Composite-Pflanzen <ol><li> Binary Konstrukte tragen Gene von Interesse wurden in A. verwandelt rhizogenes über Standardprotokolle. Wir haben eine Reihe von binären Vektoren, die das GFP-Gen als Marker, dass das Screening der transgenen Gewebe erleichtern entwickelt. Diese Vektoren enthalten verschiedene Promotoren und haben alle Gateways Klonierungsstellen (Invitrogen, Carlsbad, CA) für die Überexpression oder Inaktivierung von Genen gezielt 3,5. </li><li> A. rhizogenes ist in 50ml LB-Medium mit entsprechenden Antibiotika-Selektion bei 28 ° C für 16-24 Stunden kultiviert. </li><li> Die Bakterien werden gesammelt und zu einer endgültigen Konzentration von OD 600 resuspendiert = 0,3 in einer Stickstoff-freie Pflanze Nährlösung (Tabelle 1 und 6). </li><li> Zur Unterstützung hairy root Regeneration, eine sterilisierte Trägermatrix (&quot;Rock Wolle&quot;, Hummert International, Earth City, MO) in Stecker von ca. 3 cm 3 geschnitten. Wir legen 4 Stecker in eine Petrischale. </li><li> Ein Loch wird oben auf jeder Stecker mit einer Pipettenspitze stieß auf das Einfügen von Explantaten zu erleichtern. A. rhizogenes (5 ml) wird zu jedem Plug aufgenommen. </li><li> A schießen Abschnitt mit 2-3 Achselknospen wird durch einen schrägen Schnitt herausgeschnitten. Das Explantat wird dann in den Stecker eingeführt und angebaut bei 22 ° C für etwa drei Wochen. Wir halten die Medicago Explantate ohne Bewässerung für die ersten 10 Tage, dann, Wasser sie mit 5 ml Stickstoff-freie Lösung, wenn nötig. Wir fanden, dass das Entfernen der Sprossmeristem könnte die Bildung von Adventivwurzeln zu verringern. </li></ol> 3. Symbiontischen Rhizobien (Sinorhizobium meliloti) Infektion <ol><li> Nach drei Wochen werden einige Adventivwurzeln aus der Steinwolle entstehen. Die hairy root Composite Pflanzen sind Sand oder Vermiculit (sterilisiert) übertragen, mit oder ohne die Steinwolle. Sie sind im Gewächshaus bei 22 ° C, 16 / 8 h Licht / Dunkel-Zyklus, und 300 μmol.m -2. S -1 Lichtintensität für eine weitere Woche. </li><li> Vor Rhizobien-Infektion, S. meliloti 1201 ist in 50 ml Hefeextrakt-Mannitol-Medium für 7 Tage bei 30 ° C (Tabelle 2, und 6,7) kultiviert. </li><li> Re suspendiert Rhizobien Zellen sind, um eine endgültige Konzentration von OD 600 = 0,08 unter Verwendung von Stickstoff frei Ernährung Lösung verdünnt. Ein 10-ml frischen Suspension von S. meliloti wurde jedem Verbund-Anlage angewendet, um Knoten zu induzieren. </li></ol> 4. Transgene Root-Identification <ol><li> Zwei Wochen nach Rhizobien Inokulation werden die Composite-Pflanzen durch Waschen mit Wasser bis verwurzelt. Die Wurzeln können leicht von Sand oder Perlite werden in Wasser getrennt. </li><li> Wir legen den haarigen Wurzeln unter einem UV-Mikroskop (Nikon SMZ1500, Excitation 460-500nm, 505nm Dichroic, Barrier mehr als 510nm). Die transgenen Wurzeln sind GFP positiv und die Adventivwurzeln sind GFP negativ. Wir sammeln dann die Wurzeln according der GFP-Signal, und zählen Sie die Knoten-Nummern, messen Sie die Wurzellänge und berechnen lateralen Wurzel Dichte. Die GFP negativen Wurzeln als Kontrolle verwendet. </li></ol> 5. Repräsentatives Ergebnis In unserem Experiment, das neue Haarwurzeln aus den Explantaten in 2-3 Wochen regeneriert nach A. rhizogenes Inokulation. Unter dem UV-Mikroskop zeigen transgene Wurzeln Durchführung der GFP-Gen starke grüne Fluoreszenz (Abb. 1). Die Höhe der regeneriert Wurzeln und der Anteil der GFP positiven Wurzeln hängen von den Bedingungen der Explantate und das Wachstum Umgebung von Composite plants.On Durchschnitt waren 25% der Wurzeln produziert transgenen haarigen Roots 3. Um die Transformationseffizienz, 1) zu erhöhen die Kunststoffabdeckung doom, wie auf dem Video zu sehen, genügt, um die Feuchtigkeit in das Wachstum Fach für die ersten paar Tage zu halten und Pflanzen benötigen nur wenig Wasser in den ersten Tagen. Übermäßige Bewässerung ist schädlich für hairy root Bildung, 2) die Konzentration von Agrobacterium innoculant ist für die Transformation von Bedeutung. Übermäßige Menge an Zellen sind nicht hilfreich, hairy root Bildung oder Knotenbildung. Für Knotenbildung, die Bewässerung Lösung zu Stickstoff-free Pakete braucht, sonst werden nur wenige Knötchen bilden. Die hairy root Composite Pflanzen lassen sich mit den Keimblättern bzw. entact Sämlinge als Ausgangsmaterial werden. Nur geringfügige Änderungen der oben genannten Protokoll sind ncessary zu haarigen Wurzeln aus anderen Geweben 8 zu erzeugen. Wichtig ist, dass jeder hairy root ein unabhängiges Transformationsereignis. Daher sind die Phänotypen in einem Composite-Anlage beobachtet die Summe von mehreren transforamtion Ereignisse, die durch Wiederholungen in mehreren zusammengesetzten Pflanzen bestätigt werden benötigt <img src="/files/ftp_upload/2633/2633fig1.jpg" alt="Abbildung 1" /> Abbildung 1. A. Transgene Wurzeln aus haarigen Wurzeln sortiert werden. B. Knöllchen wurden in der behaarten Wurzel Composite Pflanzen gebildet. Wir setzten 4-Wochen alten M. truncatula hairy root Pflanzen unter dem UV-Mikroskop und dem GFP positive Wurzeln können leicht identifiziert werden. (Roter Pfeil: GFP Wurzel; Gelber Pfeil: non-GFP root) <table border="1"><tbody><tr><td> Lager </td><td> g/200ml </td><td> ml Brühe / Liter </td></tr><tr><td> MgSO 4 .7 H 2 O </td><td> 12,3 </td><td> 2 </td></tr><tr><td> CaCl 2 .2 H 2 O </td><td> 14,7 </td><td> 4 </td></tr><tr><td> K 2 HPO 4 .3 H 2 O </td><td> 6,8 </td><td> 1 </td></tr><tr><td> K 2 SO 4 </td><td> 11 </td><td> 4 </td></tr><tr><td> Fe Cl 3 .6 H 2 O </td><td> 0,49 </td><td> 2,5 </td></tr><tr><td> Mikronährstoffe </td><td> Siehe unten </td><td> 1 </td></tr></tbody></table><table border="1"><tbody><tr><td> Mikronährstoffe </td><td> g pro Liter </td></tr><tr><td> H 3 BO 3 </td><td> 0,142 </td></tr><tr><td> MnSO 4. H 2 O </td><td> 0,077 </td></tr><tr><td> ZnSO 4 .7 H 2 O </td><td> 0,1725 </td></tr><tr><td> CuSO 4 .5 H 2 O </td><td> 0,037 </td></tr><tr><td> NaMoO 4. H 2 O </td><td> 0,024 </td></tr><tr><td> CoCl 2. H 2 O </td><td> 0,0025 </td></tr><tr><td> NiSO 4 </td><td> 0,001 </td></tr></tbody></table> Tabelle 1. Nitogen-freie Ernährung Lösung <table border="1"><tbody><tr><td> K 2 HPO4 </td><td> 0.5g </td></tr><tr><td> NaCl </td><td> 0.1g </td></tr><tr><td> MgSO 4 · 7H 2 O </td><td> 0.2g </td></tr><tr><td> Hefeextrakt </td><td> 0,4 g </td></tr><tr><td> Mannitol </td><td> 10g </td></tr><tr><td> pH = 6,8 </td><td></td></tr></tbody></table> Tabelle 2. Hefeextrakt Mannitol-Medium (pro Liter)

<ol>
	<li>Limpens, E., Ramos, J., Franken, C., Raz, V., Compaan, B., Franssen, H., Bisseling, T., Geurts, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNA+interference+in+Agrobacterium+rhizogenes-transformed+roots+of+Arabidopsis+and+Medicago+truncatula.">RNA interference in Agrobacterium rhizogenes-transformed roots of Arabidopsis and Medicago truncatula.</a> J. Exp. Bot. 55, 983-992 (2004).</li><li>Collier, R., Fuchs, B., Walter, N., Kevin, L. W., Taylor, C. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ex+vitro+composite+plants:+an+inexpensive,+rapid+method+for+root+biology.">Ex vitro composite plants: an inexpensive, rapid method for root biology.</a> Plant J. 43, 449-457 (2005).</li><li>Zhang, J., Subramanian, S., Stacey, G., Yu, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flavones+and+flavonols+play+distinct+critical+roles+during+nodulation+of+Medicago+truncatula+by+Sinorhizobium+meliloti.">Flavones and flavonols play distinct critical roles during nodulation of Medicago truncatula by Sinorhizobium meliloti.</a> Plant J. 57, 171-183 (2009).</li><li>Boisson-Dernier, A., Chabaud, M., Garcia, F., Becard, G., Rosenberg, C., Barker, D. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Agrobacterium-rhizogenes-transformed+roots+of+Medicago+truncatula+for+the+study+of+nitrogen-fixing+and+endomycorrhizal+symbiotic+associations.">Agrobacterium-rhizogenes-transformed roots of Medicago truncatula for the study of nitrogen-fixing and endomycorrhizal symbiotic associations.</a> Mol. Plant-Microbe Interact. 14, 695-700 (2001).</li><li>Zhang, J., Subramanian, S., Zhang, Y., Yu, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flavone+synthases+from+Medicago+truncatula+are+flavanone-2-hydroxylases+and+are+important+for+nodulation.">Flavone synthases from Medicago truncatula are flavanone-2-hydroxylases and are important for nodulation.</a> Plant Physiol. 144, 741-751 .</li><li>Subramanian, S., Hu, X., Lu, G., Odelland, J., Yu, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+promoters+of+two+isoflavone+synthase+genes+respond+differentially+to+nodulation+and+defense+signals+in+transgenic+soybean+roots.">The promoters of two isoflavone synthase genes respond differentially to nodulation and defense signals in transgenic soybean roots.</a> Plant Mol Biol. 54, 623-639 (2004).</li><li>Vincent, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> A manual for the practical study of root nodule bacteria International Biological Program Handbook. , 1-13 (1970).</li><li>Subramanian, S., Graham, M. Y., Yu, O., Graham, T. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNA+Interference+of+Soybean+Isoflavone+Synthase+Genes+Leads+to+Silencing+in+Tissues+Distal+to+the+Transformation+Site+and+to+Enhanced+Susceptibility+to+Phytophthora+sojae.">RNA Interference of Soybean Isoflavone Synthase Genes Leads to Silencing in Tissues Distal to the Transformation Site and to Enhanced Susceptibility to Phytophthora sojae.</a> Plant Physiol. 137, 1345-1353 (2005).</li></ol>

Generieren hairy root Composite-Anlage ist eine schnelle und einfache Methode, um große Mengen von transgenen Material für viele dikotyle Arten zu erhalten. Obwohl diese Methode nicht selbst herstellen kann transgenen Samen, kann es transgenen Materialien in ein paar Wochen zu produzieren. Die Methode eignet sich besonders für Anlagen, die Schwierigkeit, über Gewebekulturen oder die Erzeugung von stabilen Transformanten haben. Im Laufe der Jahre haben wir diese Technologie verwendet, um Genfunktionen, Promoter Funktionen, microRNAs, Wurzel-und seitliche Wurzel Entwicklung, Verteidigung und abiotischen Stress-Reaktionen, Knotenbildung und andere symbiotische Prozesse, Hormon-Reaktionen, Metabolic Profiling, Gen-Profiling-, Proteom-Analyse und Studie andere biologische Prozesse. Das Protokoll ist robust und reproduzierbar.

generation of composite plants in medicago truncatula used for nodulation assays

Ähnlich wie Agrobacterium tumerfaciens kann Agrobacterium rhizogenes ausländischen DNAs in Pflanzenzellen auf das autonome root-induzierenden (Ri)-Plasmid basierte Übertragung. A. rhizogenes kann hairy root Bildung auf dem Pflanzengewebe und bilden zusammengesetzte Pflanzen nach der Transformation. Auf diesen Korbblütler, sind einige der Wurzeln regeneriert transgenen, trägt das Wildtyp-T-DNA und der technisch binären Vektors, während die Triebe sind noch nicht transgenen, dazu dient, Energie und Wachstum zu unterstützen. Diese hairy root Composite Pflanzen produziert nicht transgenen Samen, aber es gibt eine Reihe von wichtigen Funktionen, die diese Korbblütler sehr nützlich machen in der Pflanzenforschung. Zuerst mit einem breiten Wirtsspektrum, A. rhizogenes lassen sich viele Pflanzenarten, insbesondere Dikotyledonen, so dass die Gentechnik in einer Vielzahl von Arten. Zweitens, A. rhizogenes infizieren Gewebe und Explantate direkt, keine Gewebekulturen vor der Transformation ist notwendig, um Composite-Pflanzen zu erhalten, wodurch sie ideal für die Transformation von widerspenstigen Pflanzenarten. Darüber hinaus können transgene Wurzelgewebe in einer Angelegenheit von Wochen generiert werden. Für Medicago truncatula, können wir transgenen Wurzeln in so kurz wie 3 Wochen erhalten, schneller als normal floral dip Arabidopsis-Transformation. Insgesamt ist die hairy root Composite Anlagentechnik ein vielseitiges und nützliches Werkzeug, um Genfunktionen zu studieren und root related-Phänotypen. Hier zeigen wir, wie hairy root Composite Pflanzen verwendet werden, um Pflanzen-Interaktionen und Rhizobium nodulation in der schwer zu verwandeln Arten M. Studie werden truncatula.

Watch this Scientific Journal Video about Generation of Composite Plants in Medicago truncatula used for Nodulation Assays at JoVE.com

Generation of Composite Plants in Medicago truncatula used for Nodulation Assays

Wir zeigen, wie hairy root Composite Pflanzen können verwendet werden, um Pflanzen-Interaktionen und Rhizobium nodulation in der schwer zu verwandeln Arten zu studieren<em&gt; Medicago truncatula</em&gt;.

Generierung von Composite-Anlagen in Medicago truncatula Für Nodulation Assays verwendet

Similar to Agrobacterium tumerfaciens, Agrobacterium rhizogenes can transfer foreign DNAs into plant cells based on the autonomous root-inducing (Ri) ...

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Biology

Generation of Composite Plants in Medicago truncatula used for Nodulation Assays

15.9K Aufrufe.  Donald Danforth Plant Science Center. Wir zeigen, wie hairy root Composite Pflanzen können verwendet werden, um Pflanzen-Interaktionen und Rhizobium nodulation in der schwer zu verwandeln Arten zu studieren Medicago truncatula.

Serie: Generation of Composite Plants in Medicago truncatula used for Nodulation Assays

Neurocircuit Assays for Seizures in Epilepsy Mutants of Drosophila

Drosophila melanogaster is a useful tool for studying seizure like activity. A variety of mutants in which seizures can be induced through either physical shock or electrical stimulation is available for study of various aspects of seizure activity and behavior. All flies, including wild-type, will undergo seizure-like activity if stimulated at a high enough voltage. Seizure like activity is an all-or-nothing response and each genotype has a specific seizure threshold. The seizure threshold of a specific genotype of fly can be altered either by treatment with a drug or by genetic suppression or enhancement. The threshold is easily measured by electrophysiology. Seizure-like activity can be induced via high frequency electrical stimulation delivered directly to the brain and recorded through the dorsal longitudinal muscles (DLMs) in the thorax. The DLMs are innervated by part of the giant fiber system. Starting with low voltage, high frequency stimulation, and subsequently raising the voltage in small increments, the seizure threshold for a single fly can be measured.

Neurocircuit Assays for Seizures in Epilepsy Mutants of Drosophila

Using high frequency electrical stimulation, seizure-like activity can be induced in Drosophila. This activity is easily recorded from the giant fiber system.

Neurocircuit Assays für Anfällen bei Epilepsie Mutanten Drosophila</em

Drosophila melanogaster is a useful tool for studying seizure like activity. A variety of mutants in which seizures can be induced through either physical ...

Forschung

Biologie

Analysis of Schwann-astrocyte Interactions Using In Vitro Assays

Schwann cells are one of the commonly used cells in repair strategies following spinal cord injuries. Schwann cells are capable of supporting axonal regeneration and sprouting by secreting growth factors 1,2 and providing growth promoting adhesion molecules 3 and extracellular matrix molecules 4. In addition they myelinate the demyelinated axons at the site of injury 5.
However following transplantation, Schwann cells do not migrate from the site of implant and do not intermingle with the host astrocytes 6,7. This results in formation of a sharp boundary between the Schwann cells and astrocytes, creating an obstacle for growing axons trying to exit the graft back into the host tissue proximally and distally. Astrocytes in contact with Schwann cells also undergo hypertrophy and up-regulate the inhibitory molecules 8-13.
In vitro assays have been used to model Schwann cell-astrocyte interactions and have been important in understanding the mechanism underlying the cellular behaviour.
These in vitro assays include boundary assay, where a co-culture is made using two different cells with each cell type occupying different territories with only a small gap separating the two cell fronts. As the cells divide and migrate, the two cellular fronts get closer to each other and finally collide. This allows the behaviour of the two cellular populations to be analyzed at the boundary. Another variation of the same technique is to mix the two cellular populations in culture and over time the two cell types segregate with Schwann cells clumped together as islands in between astrocytes together creating multiple Schwann-astrocyte boundaries.
The second assay used in studying the interaction of two cell types is the migration assay where cellular movement can be tracked on the surface of the other cell type monolayer 14,15. This assay is commonly known as inverted coverslip assay. Schwann cells are cultured on small glass fragments and they are inverted face down onto the surface of astrocyte monolayers and migration is assessed from the edge of coverslip.
Both assays have been instrumental in studying the underlying mechanisms involved in the cellular exclusion and boundary formation. Some of the molecules identified using these techniques include N-Cadherins 15, Chondroitin Sulphate proteoglycans(CSPGs) 16,17, FGF/Heparin 18, Eph/Ephrins19.
This article intends to describe boundary assay and migration assay in stepwise fashion and elucidate the possible technical problems that might occur.

Analysis of Schwann-astrocyte Interactions Using In Vitro Assays

This article intends to describe in stepwise fashion the commonly used in vitro assays used in studying Schwann cell-asrtocyte interactions.

Analyse von Schwann-Astrozyten Wechselwirkungen mit In Vitro Assays

Schwann cells are one of the commonly used cells in repair strategies following spinal cord injuries. Schwann cells are capable of supporting axonal ...

In Situ Hybridization for the Precise Localization of Transcripts in Plants

With the advances in genomics research of the past decade, plant biology has seen numerous studies presenting large-scale quantitative analyses of gene expression. Microarray and next generation sequencing approaches are being used to investigate developmental, physiological and stress response processes, dissect epigenetic and small RNA pathways, and build large gene regulatory networks1-3. While these techniques facilitate the simultaneous analysis of large gene sets, they typically provide a very limited spatiotemporal resolution of gene expression changes. This limitation can be partially overcome by using either profiling method in conjunction with lasermicrodissection or fluorescence-activated cell sorting4-7. However, to fully understand the biological role of a gene, knowledge of its spatiotemporal pattern of expression at a cellular resolution is essential. Particularly, when studying development or the effects of environmental stimuli and mutants can the detailed analysis of a gene's expression pattern become essential. For instance, subtle quantitative differences in the expression levels of key regulatory genes can lead to dramatic phenotypes when associated with the loss or gain of expression in specific cell types. 
Several methods are routinely used for the detailed examination of gene expression patterns. One is through analysis of transgenic reporter lines. Such analysis can, however, become time-consuming when analyzing multiple genes or working in plants recalcitrant to transformation. Moreover, an independent validation to ensure that the transgene expression pattern mimics that of the endogenous gene is typically required. Immunohistochemical protein localization or mRNA in situ hybridization present relatively fast alternatives for the direct visualization of gene expression within cells and tissues. The latter has the distinct advantage that it can be readily used on any gene of interest. In situ hybridization allows detection of target mRNAs in cells by hybridization with a labeled anti-sense RNA probe obtained by in vitro transcription of the gene of interest. 
Here we outline a protocol for the in situ localization of gene expression in plants that is highly sensitivity and specific. It is optimized for use with paraformaldehyde fixed, paraffin-embedded sections, which give excellent preservation of histology, and DIG-labeled probes that are visualized by immuno-detection and alkaline-phosphatase colorimetric reaction. This protocol has been successfully applied to a number of tissues from a wide range of plant species, and can be used to analyze expression of mRNAs as well as small RNAs8-14.

In Situ Hybridization for the Precise Localization of Transcripts in Plants

The in situ hybridization protocol described here allows a direct localization of mRNA and small RNA expression at the cellular level with high sensitivity and specificity. The procedure is optimized for paraffin-embedded plant tissue sections, is applicable to a wide range of plants and tissues, and can be completed within ten days.

In-situ-Hybridisierung für die genaue Lokalisierung der Transkripte in Pflanzen

With the advances in genomics research of the past decade, plant biology has seen numerous studies presenting large-scale quantitative analyses of gene ...

Viability Assays for Cells in Culture

Manual cell counts on a microscope are a sensitive means of assessing cellular viability but are time-consuming and therefore expensive. Computerized viability assays are expensive in terms of equipment but can be faster and more objective than manual cell counts. The present report describes the use of three such viability assays. Two of these assays are infrared and one is luminescent. Both infrared assays rely on a 16&#160;bit Odyssey Imager. One infrared assay uses the DRAQ5 stain for nuclei combined with the Sapphire stain for cytosol and is visualized in the 700 nm channel. The other infrared assay, an In-Cell Western, uses antibodies against cytoskeletal proteins (&#945;-tubulin or microtubule associated protein 2) and labels them in the 800 nm channel. The third viability assay is a commonly used luminescent assay for ATP, but we use a quarter of the recommended volume to save on cost. These measurements are all linear and correlate with the number of cells plated, but vary in sensitivity. All three assays circumvent time-consuming microscopy and sample the entire well, thereby reducing sampling error. Finally, all of the assays can easily be completed within one day of the end of the experiment, allowing greater numbers of experiments to be performed within short timeframes. However, they all rely on the assumption that cell numbers remain in proportion to signal strength after treatments, an assumption that is sometimes not met, especially for cellular ATP. Furthermore, if cells increase or decrease in size after treatment, this might affect signal strength without affecting cell number. We conclude that all viability assays, including manual counts, suffer from a number of caveats, but that computerized viability assays are well worth the initial investment. Using all three assays together yields a comprehensive view of cellular structure and function.

Therapeutic compounds are often first examined in vitro with viability assays. Blind cell counts by a human observer can be highly sensitive to small changes in cell number but do not assess function. Computerized viability assays, as described here, can assess both structure and function in an objective manner.

Die Lebensfähigkeit Assays für Zellen in Kultur

Manual cell counts on a microscope are a sensitive means of assessing cellular viability but are time-consuming and therefore expensive. Computerized ...

Efficient Generation Human Induced Pluripotent Stem Cells from Human Somatic Cells with Sendai-virus

A few years ago, the establishment of human induced pluripotent stem cells (iPSCs) ushered in a new era in biomedicine. Potential uses of human iPSCs include modeling pathogenesis of human genetic diseases, autologous cell therapy after gene correction, and personalized drug screening by providing a source of patient-specific and symptom relevant cells. However, there are several hurdles to overcome, such as eliminating the remaining reprogramming factor transgene expression after human iPSCs production. More importantly, residual transgene expression in undifferentiated human iPSCs could hamper proper differentiations and misguide the interpretation of disease-relevant in vitro phenotypes. With this reason, integration-free and/or transgene-free human iPSCs have been developed using several methods, such as adenovirus, the piggyBac system, minicircle vector, episomal vectors, direct protein delivery and synthesized mRNA. However, efficiency of reprogramming using integration-free methods is quite low in most cases.
Here, we present a method to isolate human iPSCs by using Sendai-virus (RNA virus) based reprogramming system. This reprogramming method shows consistent results and high efficiency in cost-effective manner.

Here, we present our established method to reprogram human somatic cells into transgene-free human iPSCs with Sendai virus, which shows consistent outcome and enhanced efficiency.

Effiziente Erzeugung Menschen induzierten pluripotenten Stammzellen aus menschlichen Körperzellen mit Sendai-Virus

A few years ago, the establishment of human induced pluripotent stem cells (iPSCs) ushered in a new era in biomedicine. Potential uses of human iPSCs ...

MALDI-Mass Spectrometric Imaging for the Investigation of Metabolites in Medicago truncatula Root Nodules

Most techniques used to study small molecules, such as pharmaceutical drugs or endogenous metabolites, employ tissue extracts which require the homogenization of the tissue of interest that could potentially cause changes in the metabolic pathways being studied1. Mass spectrometric imaging (MSI) is a powerful analytical tool that can provide spatial information of analytes within intact slices of biological tissue samples1-5. This technique has been used extensively to study various types of compounds including proteins, peptides, lipids, and small molecules such as endogenous metabolites. With matrix-assisted laser desorption/ionization (MALDI)-MSI, spatial distributions of multiple metabolites can be simultaneously detected. Herein, a method developed specifically for conducting untargeted metabolomics MSI experiments on legume roots and root nodules is presented which could reveal insights into the biological processes taking place. The method presented here shows a typical MSI workflow, from sample preparation to image acquisition, and focuses on the matrix application step, demonstrating several matrix application techniques that are useful for detecting small molecules. Once the MS images are generated, the analysis and identification of metabolites of interest is discussed and demonstrated. The standard workflow presented here can be easily modified for different tissue types, molecular species, and instrumentation.

MALDI-Mass Spectrometric Imaging for the Investigation of Metabolites in Medicago truncatula Root Nodules

Mass spectrometric imaging (MSI) is a powerful tool that can be used to discover and identify various chemical species in intact tissues, preserving the compounds in their native environments, which can provide new insights into biological processes. Herein a MSI method developed for the analysis of small molecules is described.&#160;

MALDI-Imaging-Massenspektrometrie zur Untersuchung von Metaboliten in<em&gt; Medicago truncatula</em&gt; Wurzelknöllchen

Most techniques used to study small molecules, such as pharmaceutical drugs or endogenous metabolites, employ tissue extracts which require the ...

Measuring Spatial and Temporal Ca2+ Signals in Arabidopsis Plants

Developmental and environmental cues induce Ca2+ fluctuations in plant cells. Stimulus-specific spatial-temporal Ca2+ patterns are sensed by cellular Ca2+ binding proteins that initiate Ca2+ signaling cascades. However, we still know little about how stimulus specific Ca2+ signals are generated. The specificity of a Ca2+ signal may be attributed to the sophisticated regulation of the activities of Ca2+ channels and/or transporters in response to a given stimulus. To identify these cellular components and understand their functions, it is crucial to use systems that allow a sensitive and robust recording of Ca2+ signals at both the tissue and cellular levels. Genetically encoded Ca2+ indicators that are targeted to different cellular compartments have provided a platform for live cell confocal imaging of cellular Ca2+ signals. Here we describe instructions for the use of two Ca2+ detection systems: aequorin based FAS (film adhesive seedlings) luminescence Ca2+ imaging and case12 based live cell confocal fluorescence Ca2+ imaging. Luminescence imaging using the FAS system provides a simple, robust and sensitive detection of spatial and temporal Ca2+ signals at the tissue level, while live cell confocal imaging using Case12 provides simultaneous detection of cytosolic and nuclear Ca2+ signals at a high resolution.

Measuring Spatial and Temporal Ca2+ Signals in Arabidopsis Plants

Ca2+ signaling regulates diverse biological processes in plants. Here we present approaches for monitoring abiotic stress induced spatial and temporal Ca2+ signals in Arabidopsis cells and tissues using the genetically encoded Ca2+ indicators Aequorin or Case12.

Mess räumliche und zeitliche Ca<sup&gt; 2 +</sup&gt; Signale in Arabidopsis-Pflanzen

Developmental and environmental cues induce Ca2+ fluctuations in plant cells. Stimulus-specific spatial-temporal Ca2+ patterns are sensed by cellular Ca2+ ...

MRM Microcoil Performance Calibration and Usage Demonstrated on Medicago truncatula Roots at 22 T

This protocol describes a signal-to-noise ratio (SNR) calibration and sample preparation method for solenoidal microcoils combined with biological samples, designed for high-resolution magnetic resonance imaging (MRI), also referred to as MR microscopy (MRM). It may be used at pre-clinical MRI spectrometers, demonstrated on Medicago truncatula root samples. Microcoils increase sensitivity by matching the size of the RF resonator to the size of the sample of interest, thereby enabling higher image resolutions in a given data acquisition time. Due to the relatively simple design, solenoidal microcoils are straightforward and cheap to construct and can be easily adapted to the sample requirements. Systematically, we explain how to calibrate new or home-built microcoils, using a reference solution. The calibration steps include: pulse power determination using a nutation curve; estimation of RF-field homogeneity; and calculating a volume-normalized signal-to-noise ratio (SNR) using standard pulse sequences. Important steps in sample preparation for small biological samples are discussed, as well as possible mitigating factors such as magnetic susceptibility differences. The applications of an optimized solenoid coil are demonstrated by high-resolution (13 x 13 x 13 &#956;m3, 2.2 pL) 3D imaging of a root sample.

MRM Microcoil Performance Calibration and Usage Demonstrated on Medicago truncatula Roots at 22 T

A protocol to study biological tissue at high spatial resolution using ultra-high field magnetic resonance microscopy (MRM) using microcoils is presented. Step-by-step instructions are provided for characterizing the microcoils. Finally, optimization of imaging is demonstrated on plant roots.

MRM Microcoil Performance Calibration and Usage demonstriert auf Medicago-Truncatula-Wurzeln bei 22 T

This protocol describes a signal-to-noise ratio (SNR) calibration and sample preparation method for solenoidal microcoils combined with biological ...

Microscopy Techniques for Interpreting Fungal Colonization in Mycoheterotrophic Plants Tissues and Symbiotic Germination of Seeds

Structural botany is an indispensable perspective to fully understand the ecology, physiology, development, and evolution of plants. When researching mycoheterotrophic plants (i.e., plants that obtain carbon from fungi), remarkable aspects of their structural adaptations, the patterns of tissue colonization by fungi, and the morphoanatomy of subterranean organs can enlighten their developmental strategies and their relationships with hyphae, the source of nutrients. Another important role of symbiotic fungi is related to the germination of orchid seeds; all Orchidaceae species are mycoheterotrophic during germination and seedling stage (initial mycoheterotrophy), even the ones that photosynthesize in adult stages. Due to the lack of nutritional reserves in orchid seeds, fungal symbionts are essential to provide substrates and enable germination. Analyzing germination stages by structural perspectives can also answer important questions regarding the fungi interaction with the seeds. Different imaging techniques can be applied to unveil fungi endophytes in plant tissues, as are proposed in this article. Freehand and thin sections of plant organs can be stained and then observed using light microscopy. A fluorochrome conjugated to wheat germ agglutinin can be applied to the fungi and co-incubated with Calcofluor White to highlight plant cell walls in confocal microscopy. In addition, the methodologies of scanning and transmission electron microscopy are detailed for mycoheterotrophic orchids, and the possibilities of applying such protocols in related plants is explored. Symbiotic germination of orchid seeds (i.e., in the presence of mycorrhizal fungi) is described in the protocol in detail, along with possibilities of preparing the structures obtained from different stages of germination for analyses with light, confocal, and electron microscopy.

This protocol aims to provide detailed procedures for collecting, fixing, and maintaining mycoheterotrophic plant samples, applying different microscopy techniques such as scanning and transmission electron microscopy, light, confocal, and fluorescence microscopy to study fungal colonization in plants tissues and seeds germinated with mycorrhizal fungi.

Mikroskopische Techniken zur Interpretation der Pilzbesiedlung in mykoheterotrophen Pflanzengeweben und der symbiotischen Keimung von Samen

Structural botany is an indispensable perspective to fully understand the ecology, physiology, development, and evolution of plants. When researching ...

AirID-Based Proximity Labeling for Protein-Protein Interaction in Plants

In mammalian cells and plants, proximity labeling (PL) approaches using modified ascorbate peroxidase (APEX) or the Escherichia coli biotin ligase BirA (known as BioID) have proven successful in identifying protein-protein interactions (PPIs). APEX, BioID, and TurboID, a revised version of BioID have some restrictions in addition to being valuable technologies. The recently developed AirID, a novel version of BioID for proximity identification in protein-protein interactions, overcame these restrictions. Previously, AirID has been used in animal models, while the current study demonstrates the use of AirID in plants, and the results confirmed that AirID performs better in plant systems as compared to other PL enzymes such as BioID and TurboID for protein labeling that are proximal to the target proteins. Here is a step-by-step protocol for identifying protein interaction partners using AT4G18020 (APRR2) protein as a model. The methods describe the construction of vector, the transformation of construct through agroinfiltration, biotin transformation, extraction of proteins, and enrichment of biotin-labeled proteins through affinity purification technique. The results conclude that AirID is a novel and ideal enzyme for analyzing PPIs in plants. The method can be applied to study other proteins in plants.

Here, we present a step-by-step protocol for performing the proximity labeling (PL) experiment in cucumber (Cucumis sativus L.) using AT4G18020 (APRR2)-AirID protein as a model. The method describes the construction of a vector, the transformation of a construct through agroinfiltration, biotin infiltration, protein extraction, and purification of biotin-labeled proteins through affinity purification technique.

AirID-basierte Proximity-Markierung für Protein-Protein-Interaktion in Pflanzen

In mammalian cells and plants, proximity labeling (PL) approaches using modified ascorbate peroxidase (APEX) or the Escherichia coli biotin ligase BirA ...