Les auteurs tiennent à remercier le Dr Chris Taylor (Ohio State University) pour introduire une technologie hairy root à notre laboratoire et les Drs. Senthil Subramanian (South Dakota State University), Juan Zhang (Université Ludong, Shandong en Chine) pour améliorer le protocole. Nous remercions également le Dr Cheng Hao (Nanjing University Agriculture) pour aide à faire cette vidéo. Ce travail est soutenu en partie par des subventions de DOE (DE-SC0001295), la NSF (MCB-0923779) et l&#39;USDA (2010-65116-20514) à OY

Le protocole suivant a été utilisé pour générer des plantes à racines poilues composites dans un modèle de légumineuses espèce M. truncatula. Des protocoles similaires ont été adaptés pour au moins huit espèces végétales 1-4. Nous avons utilisé M. truncatula plantes racines chevelues composites pour étudier les fonctions des gènes dans les racines et le développement de nodules. Le protocole a été séparé en quatre sections: 1) la préparation de matières végétales; 2) générant plantes à racines poilues composites; 3) l&#39;infection symbiotique Rhizobium, et 4) l&#39;identification racines transgéniques. Nous avons utilisé le vecteur binaire contenant la protéine fluorescente verte (GFP) en tant que journaliste pour le dépistage racines transgéniques dans les plantes composites 3. Comparé aux antibiotiques à base de sélection, GFP-dépistage est rapide, facile et peu coûteux. Dans notre construction, l&#39;ER-expression de gène de la GFP optimisé est entraîné par le promoteur ubiquitine superbe, qui a de forts signaux GFP constitutive dans les racines transgéniques, permettant la distinction facile entre les racines transgéniques et non transgéniques. 1. Préparation des matériaux végétaux <ol><li> M. graines sont récoltées truncautla à partir de plantes cultivées dans une serre (50% d&#39;humidité relative, 16 / 8 h de lumière / obscurité, 22/18 ° C température jour / nuit). Les graines mûres peuvent être stockées à 4 ° C. </li><li> Environ 100 graines sont scarifiées dans 10 ml d&#39;acide sulfurique concentré (95-98%, Sigma-Aldrich, MO) pendant 10 min avec agitation périodique. </li><li> Les graines sont rincées 5-7 fois avec de l&#39;eau stérile avec une légère agitation. </li><li> Les graines sont ensuite trempées dans l&#39;eau à température ambiante pendant 6 heures et conservé à 4 ° C pendant 36-48 heures à synchroniser la germination. </li><li> Environ 20-30 graines sont réparties sur des sols humides des papiers-filtres placés dans une boîte de Pétri. Ils sont maintenus à 22 ° C, avec 16 / 8 h cycle lumière / obscurité, et 40 μmol.m -2. Intensité de la lumière s -1, pendant 5-6 jours. </li><li> Plants germés sont transférés dans le sol et placés dans une serre pendant un mois. </li></ol> 2. Génération plantes racines chevelues Composite <ol><li> Des constructions binaires portant des gènes d&#39;intérêt ont été transformés en A. rhizogenes utilisant des protocoles standard. Nous avons conçu un ensemble de vecteurs binaires contenant le gène de la GFP comme marqueur qui facilitent le dépistage des tissus transgéniques. Ces vecteurs contiennent divers promoteurs et tous ont des sites de clonage Gateway (Invitrogen, Carlsbad, CA) pour la sur-expression ou réprimer des gènes ciblés 3,5. </li><li> A. rhizogenes est cultivée dans 50 ml de milieu LB avec sélection antibiotique approprié à 28 ° C pendant 16-24 heures. </li><li> Les bactéries sont collectées et remises en suspension à une concentration finale de DO600 = 0,3 dans un exempt d&#39;azote des plantes des nutriments solution (tableau 1 et 6). </li><li> Pour soutenir la régénération hairy root, une matrice stérilisés à l&#39;appui (&quot;laine de roche&quot;, Hummert International, Earth City, MO) est découpé en fiches d&#39;environ 3 cm 3. Nous mettons 4 bouchons dans une boîte de Pétri. </li><li> Un trou est fourré sur le dessus de chaque fiche à l&#39;aide d&#39;une pipette pour faciliter l&#39;insertion des explants. A. rhizogenes (5 ml) est ajoutée à chaque fiche. </li><li> Une section tournage avec 2-3 bourgeons axillaires est excisé par une coupe oblique. L&#39;explant est ensuite insérée dans la prise, et cultivés à 22 ° C pendant environ trois semaines. Nous gardons les explants Medicago sans arrosage pour les 10 premiers jours, puis, arrosez-les avec 5 ml d&#39;azote sans solution lorsque nécessaire. Nous avons trouvé que la suppression du méristème apical pourrait diminuer la formation de racines adventives. </li></ol> 3. Symbiotique Rhizobium (Sinorhizobium meliloti) Infection <ol><li> Après trois semaines, quelques racines adventives ressortiront de la laine de roche. Les plantes à racines poilues composites sont transférés à du sable ou de la vermiculite (stérilisée), avec ou sans la laine de roche. Elles sont cultivées en serre à 22 ° C, 16 / 8 h cycle lumière / obscurité, et 300 μmol.m -2. S -1 intensité lumineuse pour une semaine de plus. </li><li> Avant de rhizobiums infection, S. meliloti 1201 est cultivé dans 50 d&#39;extrait de levure-mannitol ml de milieu pendant 7 jours à 30 ° C (tableau 2 et 6,7). </li><li> Re-suspendre les cellules de rhizobium sont dilués à une concentration finale de 600 OD = 0,08 solution en utilisant la nutrition azotée libre. Une suspension de 10 ml fraîche de S. meliloti a été appliquée à chaque usine composites pour induire la formation de nodules. </li></ol> 4. Identification des racines transgéniques <ol><li> Deux semaines après l&#39;inoculation de rhizobium, les plantes composites sont déracinés par un lavage à l&#39;eau. Les racines peuvent être facilement séparés de sables ou de la perlite dans l&#39;eau. </li><li> Nous mettons les racines poilues sous un microscope à UV (Nikon SMZ1500, excitation 460-500nm, 505nm dichroïque, Barrier plus de 510nm). Les racines transgéniques sont GFP-positives et les racines adventives sont GFP négative. Nous avons ensuite recueillir les racines according le signal GFP, et de compter le nombre de nodules, mesurer la longueur des racines, et de calculer la densité des racines latérales. Les racines sont négatives GFP utilisé comme témoin. </li></ol> 5. Résultat Représentant Dans notre expérience, les racines des cheveux nouveaux régénérées à partir des explants en 2-3 semaines après A. rhizogenes inoculation. Sous le microscope à UV, les racines transgéniques portant le gène de la GFP montrent une forte fluorescence verte (Fig 1). Le montant de racines et la partie régénérée des racines GFP-positives dépendent des conditions d&#39;explants et de l&#39;environnement de croissance de plants.On moyenne composite, 25% des racines produites étaient transgéniques racines poilues 3. Afin d&#39;augmenter l&#39;efficacité de transformation, 1) le destin couvercle en plastique, comme le montre la vidéo, est suffisant pour maintenir l&#39;humidité dans le bac de croissance pour les premiers jours, et les plantes ne nécessitent peu d&#39;eau dans les premiers jours. Un arrosage excessif est préjudiciable à la formation des racines velues; 2) la concentration de l&#39;inoculant Agrobacterium est important pour la transformation. Une quantité excessive de cellules ne sont pas utiles à la formation des racines velues ou la formation de nodules. Pour la formation de nodules, la solution d&#39;arrosage doit être exempt d&#39;azote, sinon, quelques nodules se forment. Les plantes à racines poilues composites peuvent être générés en utilisant les cotylédons ou les semis Entact comme matériau de départ. Seules des modifications mineures du protocole ci-dessus sont ncessary pour générer des racines poilues d&#39;autres tissus 8. Surtout, chaque racine velue est un événement de transformation indépendants. Par conséquent, les phénotypes observés dans une usine de composites sont la somme des événements transforamtion plusieurs devaient être confirmés par des répétitions dans de multiples plantes composites <img src="/files/ftp_upload/2633/2633fig1.jpg" alt="Figure 1" /> Figure 1. A. racines transgéniques peuvent être triés à partir des racines poilues. B. Nodules ont été formés dans les plantes racines chevelues composites. Nous avons placé quatre semaines, âgé de M. plantes truncatula hairy root sous le microscope à UV et les racines GFP-positives peuvent être facilement identifiés. (Flèche rouge: root GFP; flèche jaune: non-GFP racine) <table border="1"><tbody><tr><td> Stock </td><td> g/200ml </td><td> ml de stock / litre </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> Micronutriments </td><td> Voir ci-dessous </td><td> 1 </td></tr></tbody></table><table border="1"><tbody><tr><td> Micronutriments </td><td> g par litre </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> Tableau 1. Solution Nutrition Nitogen libre <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> Extrait de levure </td><td> 0.4g </td></tr><tr><td> Mannitol </td><td> 10g </td></tr><tr><td> pH = 6,8 </td><td></td></tr></tbody></table> Tableau 2. Extrait de levure moyennes mannitol (par litre)

<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>

Générer des plantes racines chevelues composite est une méthode rapide et facile à obtenir de grandes quantités de matériel transgénique pour de nombreuses espèces de dicotylédones. Bien que cette méthode ne peut pas produire des semences transgéniques, elle peut produire des matériaux transgéniques dans quelques semaines. La méthode est particulièrement adaptée pour les plantes qui ont des difficultés à établir des cultures de tissus ou de générer des transformants stables. Au fil des ans, nous avons utilisé cette technologie pour étudier les fonctions des gènes, les fonctions de promoteur, les microARN, la racine et le développement des racines latérales, la défense et des réponses aux stress abiotiques, la nodulation et d&#39;autres processus symbiotiques, les réponses hormonales, le profilage métabolique, le profilage des gènes, l&#39;analyse protéomique, et d&#39;autres processus biologiques. Le protocole est robuste et reproductible.

generation of composite plants in medicago truncatula used for nodulation assays

Similaire à tumerfaciens Agrobacterium Agrobacterium rhizogenes pouvez transférer des ADN étrangers dans les cellules végétales sur la base des racines autonomes induisant (Ri) de plasmide. A. rhizogenes peut causer la formation de racines velues sur les tissus végétaux et la forme des plantes composites après transformation. Sur ces plantes composites, quelques-unes des racines régénérées sont transgéniques, portant le type sauvage ADN-T et le vecteur binaire conçu, tandis que les pousses sont encore non transgéniques, et servant à fournir de l&#39;énergie et soutenir la croissance. Ces plantes à racines poilues composite ne produisent des semences transgéniques, mais il ya un certain nombre de caractéristiques importantes qui font de ces plantes en composite très utile dans la recherche végétale. Tout d&#39;abord, avec une large gamme d&#39;hôtes, A. rhizogenes peut transformer de nombreuses espèces végétales, en particulier les dicotylédones, ce qui permet au génie génétique dans une variété d&#39;espèces. Deuxièmement, A. rhizogenes infecter les tissus et les explants directement; pas des cultures de tissus avant la transformation est nécessaire pour obtenir des plantes composites, ce qui les rend idéales pour transformer les espèces végétales récalcitrants. Par ailleurs, les tissus des racines transgéniques peuvent être générés en quelques semaines. Pour Medicago truncatula, nous pouvons obtenir des racines transgéniques dans le plus court de trois semaines, plus vite que la normale floraux creux de transformation d&#39;Arabidopsis. Globalement, la technologie chevelu racinaire des plantes composites est un outil polyvalent et utile pour étudier les fonctions des gènes et la racine liée-phénotypes. Ici nous démontrons comment les plantes racines chevelues composite peut être utilisé pour étudier les interactions plante-Rhizobium et la nodulation dans la M. difficiles à transformer les espèces 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

Nous démontrons comment les plantes racines chevelues composite peut être utilisé pour étudier les interactions plante-Rhizobium et la nodulation dans les difficiles à transformer les espèces<em&gt; Medicago truncatula</em&gt;.

Génération de plantes dans les composites Medicago truncatula Utilisées pour les dosages nodulation

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 vues.  Donald Danforth Plant Science Center. Nous démontrons comment les plantes racines chevelues composite peut être utilisé pour étudier les interactions plante-Rhizobium et la nodulation dans les difficiles à transformer les espèces Medicago truncatula.

Vidéo: 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.

Les dosages des Saisies en Neurocircuit Mutants épilepsie du Drosophile</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 ...

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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 de Schwann-astrocytes interactions à l&#39;aide In Vitro Tests

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.

Hybridation in situ pour la localisation précise des transcriptions dans les plantes

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.

Les analyses de viabilité de cellules en culture

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.

Génération efficace d&#39;origine humaine souches pluripotentes à partir de cellules somatiques humaines avec 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;

Imagerie MALDI-spectrométrie de masse pour l&#39;investigation des métabolites dans<em&gt; Medicago truncatula</em&gt; nodules racinaires

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.

Mesure spatiale et temporelle Ca<sup&gt; 2 +</sup&gt; Signaux de plantes d&#39;Arabidopsis

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.

Étalonnage et utilisation des performances de microcoil MRM démontrés sur les racines de la truncatule Medicago à 22 T

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

Laser-Capture Microdissection RNA-Sequencing for Spatial and Temporal Tissue-Specific Gene Expression Analysis in Plants

The development of a complex multicellular organism is governed by distinct cell types that have different transcriptional profiles. To identify transcriptional regulatory networks that govern developmental processes it is necessary to measure the spatial and temporal gene expression profiles of these individual cell types. Therefore, insight into the spatio-temporal control of gene expression is essential to gain understanding of how biological and developmental processes are regulated. Here, we describe a laser-capture microdissection (LCM) method to isolate small number of cells from three barley embryo organs over a time-course during germination followed by transcript profiling. The method consists of tissue fixation, tissue processing, paraffin embedding, sectioning, LCM and RNA extraction followed by real-time PCR or RNA-seq. This method has enabled us to obtain spatial and temporal profiles of seed organ transcriptomes from varying numbers of cells (tens to hundreds), providing much greater tissue-specificity than typical bulk-tissue analyses. From these data we were able to define and compare transcriptional regulatory networks as well as predict candidate regulatory transcription factors for individual tissues. The method should be applicable to other plant tissues with minimal optimization.

Presented here is a protocol for laser-capture microdissection (LCM) of plant tissues. LCM is a microscopic technique for isolating areas of tissue in a contamination-free manner. The procedure includes tissue fixation, paraffin embedding, sectioning, LCM and RNA extraction. RNA is used in the downstream tissue-specific, temporally resolved analysis of transcriptomes.

Analyse de l’ARN de microdissection à capture laser pour analyse d’expression génique spécifique aux tissus spatiaux et temporels chez les plantes

The development of a complex multicellular organism is governed by distinct cell types that have different transcriptional profiles. To identify ...

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.

Techniques de microscopie pour interpréter la colonisation fongique dans les tissus végétaux mycohétérotrophes et germination symbiotique des graines

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