Gli autori desiderano ringraziare il Dr. Chris Taylor (Ohio State University) per introdurre la tecnologia peloso di root per il nostro laboratorio e Drs. Senthil Subramanian (South Dakota State University), Juan Zhang (Ludong University, Shandong in Cina) per migliorare il protocollo. Ringraziamo anche il Dott. Cheng Hao (Agricoltura Nanjing University) per la aiuta a fare questo video. Questo lavoro è sostenuto in parte da finanziamenti DOE (DE-SC0001295), NSF (MCB-0923779) e USDA (2010-65116-20514) per OY

Il seguente protocollo è stato utilizzato per generare piante radice pelosa composito in una specie di legume modello M. truncatula. Protocolli simili sono stati adattati per almeno otto specie vegetali 1-4. Abbiamo usato M. piante radice truncatula peloso composito per studiare le funzioni del gene in radice e lo sviluppo dei noduli. Il protocollo è stato suddiviso in quattro sezioni: 1) i materiali vegetali preparazione, 2) impianti di generazione radice pelosa compositi; 3) simbiotica Rhizobium infezione e 4) l&#39;identificazione radice transgenici. Abbiamo utilizzato il vettore binario contenente la proteina verde fluorescente (GFP) gene come reporter per lo screening radici nelle piante transgeniche composito 3. Rispetto agli antibiotici a base di selezione, GFP-screening è veloce, facile e poco costoso. Nel nostro costrutto, la ER-espressione del gene GFP-ottimizzato è guidata dal promotore super-ubiquitina, che ha forti segnali GFP costitutivo nelle radici transgenici, che permette di distinguere agevolmente le radici transgeniche e non transgeniche. 1. Preparazione Piante <ol><li> M. semi truncautla sono raccolti da piante coltivate in una serra (50% di umidità relativa, 16 / 8 h luce / buio, 22/18 ° C giorno / notte temperatura). Semi maturi possono essere conservati a 4 ° C. </li><li> Circa 100 semi sono scarificati in 10 ml di acido solforico concentrato (95-98%, Sigma-Aldrich, MO) per 10 minuti con agitazione periodica. </li><li> I semi sono 5-7 volte risciacquati con acqua sterile e scuotendo con cautela. </li><li> I semi vengono poi immersi in acqua a temperatura ambiente per 6 ore e conservato a 4 ° C per 36-48 ore per sincronizzare la germinazione. </li><li> Circa il 20-30 semi sono diffuse su suoli umidi carte da filtro posto in una capsula di Petri. Essi sono mantenuti a 22 ° C, con 16 / 8 h ciclo luce / buio, e 40 μmol.m -2. S -1 intensità della luce, per 5-6 giorni. </li><li> Piantine germinate vengono trasferiti nel terreno e messo in serra per un mese. </li></ol> 2. Generazione Piante Root Peloso Composite <ol><li> Costruisce binari che trasportano i geni di interesse sono state trasformate in A. rhizogenes utilizzando protocolli standard. Abbiamo progettato una serie di vettori binari contenenti il gene GFP come marcatore che facilitano il controllo dei tessuti transgenici. Questi vettori contengono vari promotori e tutti hanno siti clonazione Gateway (Invitrogen, Carlsbad, CA) per la sovra-espressione o silenziamento dei geni mirati 3,5. </li><li> Rhizogenes A. è coltivato in 50 ml di media LB con un&#39;appropriata selezione antibiotica a 28 ° C per 16-24 ore. </li><li> I batteri sono raccolte e risospese ad una concentrazione finale di 600 OD = 0.3 in una soluzione priva di impianto di azoto nutriente (Tabella 1 e 6). </li><li> Per sostenere la rigenerazione peloso radice, una matrice di supporto sterilizzato (&quot;Lana di Roccia&quot;, Hummert internazionale, Earth City, MO), viene tagliato in tasselli di circa 3 cm 3. Abbiamo posto 4 spine in una piastra di Petri. </li><li> Un buco è colpito sulla parte superiore di ogni spina con un puntale per facilitare l&#39;inserimento di espianti. A. rhizogenes (5 ml) viene aggiunto a ogni spina. </li><li> Una sezione sparare con 2-3 gemme ascellari è asportato da un taglio obliquo. L&#39;espianto viene quindi inserito nella presa, e cresciuto a 22 ° C per circa tre settimane. Manteniamo la Medicago espianti senza irrigazione per i primi 10 giorni, poi, l&#39;acqua con 5 ml di azoto senza soluzione quando necessario. Abbiamo trovato che la rimozione del meristema apicale potrebbe diminuire la formazione di radici avventizie. </li></ol> 3. Simbiotica Rhizobium (Sinorhizobium meliloti) Infezione <ol><li> Dopo tre settimane, alcune radici avventizie uscirà dalla lana di roccia. Le piante radice pelosa compositi vengono trasferiti in sabbia o vermiculite (sterilizzato), con o senza la lana di roccia. Esse vengono coltivate in serra a 22 ° C, 16 / 8 h ciclo luce / buio, e 300 μmol.m -2. S -1 intensità della luce per una settimana di più. </li><li> Prima di rizobi ​​infezione, S. meliloti 1201 è colto in 50 ml di estratto di lievito-mannitolo medio per 7 giorni a 30 ° C (tabella 2 e 6,7). </li><li> Risospese cellule rizobi ​​sono diluiti ad una concentrazione finale di 600 OD = 0.08 usando la soluzione di nutrizione azoto libero. A 10 ml sospensione fresca di S. meliloti è stato applicato a ciascun impianto composito di indurre la formazione di noduli. </li></ol> 4. Radice di identificazione transgenici <ol><li> Due settimane dopo l&#39;inoculazione rizobi, le piante composite sono sradicate dal lavaggio in acqua. Le radici possono essere facilmente separato dalla sabbia o perlite in acqua. </li><li> Abbiamo posto le radici peloso sotto un microscopio a raggi UV (Nikon SMZ1500, eccitazione 460-500nm, 505nm dicroico, Barriera oltre 510nm). Le radici sono transgenici GFP positive e le radici avventizie sono GFP negativi. Abbiamo poi raccogliere le radici according il segnale GFP, e contare il numero di noduli, misurare la lunghezza radice, e calcolare la densità laterale root. Le radici GFP negativi sono utilizzati come controllo. </li></ol> 5. Rappresentante dei risultati Nel nostro esperimento, le radici dei capelli nuova rigenerata dal espianti in 2-3 settimane dopo la A. rhizogenes inoculazione. Sotto il microscopio a raggi UV, le radici transgenici portatori del gene GFP mostrano una forte fluorescenza verde (Fig. 1). La quantità di radici rigenerate e la parte delle radici GFP positive dipendono dalle condizioni di espianti e l&#39;ambiente di crescita composito plants.On media, il 25% delle radici prodotte erano transgenici radici pelose 3. Per aumentare l&#39;efficienza della trasformazione, 1) il destino coperchio di plastica, come mostrato nel video, è sufficiente per mantenere l&#39;umidità nel cassetto crescita per i primi giorni, e solo piante richiedono poca acqua nei primi giorni. Irrigazione eccessiva è dannosa per la formazione delle radici pelose, 2) la concentrazione di innoculant Agrobacterium è importante per la trasformazione. Quantità eccessiva di cellule non sono utili alla formazione delle radici pelose o formazione di noduli. Per la formazione di noduli, la soluzione di irrigazione deve essere azoto-free, altrimenti, noduli di pochi forma. Le piante radice pelosa composito può essere generato usando cotiledoni o piantine Entact come materiale di partenza. Solo piccole modifiche del protocollo di cui sopra sono ncessary per generare radici pelose da altri tessuti 8. È importante sottolineare che ogni radice peloso è un evento di trasformazione indipendente. Pertanto, i fenotipi osservati in un impianto di compositi sono la somma di eventi diversi transforamtion che doveva essere confermata da ripetizioni in diverse piante composite <img src="/files/ftp_upload/2633/2633fig1.jpg" alt="Figura 1" /> Figura 1. A. radici transgeniche può essere risolto dalle radici pelose. Noduli B. si sono formati negli impianti radice pelosa composito. Abbiamo messo quattro settimane di età M. piante radice truncatula peloso sotto il microscopio a raggi UV e alle radici GFP positive possono essere facilmente identificati. (Freccia rossa: root GFP; freccia gialla: non GFP root) <table border="1"><tbody><tr><td> Stock </td><td> g/200ml </td><td> ml di brodo / litro </td></tr><tr><td> MgSO 4 0,7 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> Micronutrienti </td><td> Vedi sotto </td><td> 1 </td></tr></tbody></table><table border="1"><tbody><tr><td> Micronutrienti </td><td> g per litro </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 0,7 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> Tabella 1. Nitogen senza soluzione di nutrizione <table border="1"><tbody><tr><td> K 2 HPO4 </td><td> 0,5 g </td></tr><tr><td> NaCl </td><td> 0.1g </td></tr><tr><td> MgSO 4 · 7H 2 O </td><td> 0,2 g </td></tr><tr><td> Estratto di Lievito </td><td> 0.4g </td></tr><tr><td> Mannitolo </td><td> 10g </td></tr><tr><td> pH = 6.8 </td><td></td></tr></tbody></table> Tabella 2. Estratto di lievito medio mannitolo (per litro)

<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>

Nessun conflitto di interessi dichiarati.

Generazione impianto peloso radice composito è un metodo rapido e semplice per ottenere grandi quantità di materiale transgenico per molte specie dicot. Anche se questo metodo non è possibile produrre sementi transgeniche, può produrre materiale transgenico in poche settimane. Il metodo è particolarmente adatto per le piante che hanno difficoltà a stabilire colture di tessuti o generare trasformanti stabili. Nel corso degli anni, abbiamo utilizzato questa tecnologia per studiare le funzioni del gene, le funzioni di promotore, microRNA, radice e lo sviluppo delle radici laterali, difesa e le risposte allo stress abiotici, nodulazione e altri processi simbiotici, le risposte ormonali, profili metabolici, profilazione dell&#39;espressione genica, analisi proteomica, e altri processi biologici. Il protocollo è robusto e replicabile.

generation of composite plants in medicago truncatula used for nodulation assays

Simile a tumerfaciens Agrobacterium, rhizogenes Agrobacterium può trasferire DNA estraneo nelle cellule vegetali in base alla radice autonomo che induce (Ri) plasmide. A. rhizogenes può causare la formazione pelosa root su tessuti vegetali e piante composite forma dopo la trasformazione. Su queste piante composite, alcune delle radici rigenerate sono transgenici, portando il tipo selvatico T-DNA e il vettore binario progettato, mentre i germogli sono ancora non transgenico, serve a fornire energia e sostenere la crescita. Queste piante radice pelosa composito non producono semi transgenici, ma ci sono una serie di importanti caratteristiche che rendono queste piante composite molto utile nella ricerca delle piante. In primo luogo, con una vasta gamma degli ospiti, A. rhizogenes può trasformare molte specie di piante, soprattutto dicotiledoni, permettendo l&#39;ingegneria genetica in una varietà di specie. In secondo luogo, A. rhizogenes infettare i tessuti e espianti direttamente, senza colture di tessuti prima trasformazione è necessaria per ottenere piante composite, che li rende ideali per trasformare specie vegetali recalcitranti. Inoltre, i tessuti radice transgenico può essere generato in poche settimane. Per Medicago truncatula, possiamo ottenere radici transgenico nel più breve di tre settimane, più velocemente del normale floreali tuffo trasformazione di Arabidopsis. Nel complesso, il peloso tecnologia dell&#39;impianto radice composito è uno strumento versatile e utile per studiare le funzioni dei geni e radice-fenotipi correlati. Qui mostriamo come le piante radice pelosa compositi possono essere utilizzati per studiare le interazioni pianta-Rhizobium e nodulazione nelle difficili da trasformare la specie M. 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

Si dimostra come le piante radice pelosa compositi possono essere utilizzati per studiare le interazioni pianta-Rhizobium e nodulazione nelle difficili da trasformare la specie<em&gt; Medicago truncatula</em&gt;.

Generazione di piante Composite in Medicago truncatula Per saggi nodulazione

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

generation-composite-plants-medicago-truncatula-used-for-nodulation

Nanyang Technological University

Research

JoVE Journal

Biology

Generation of Composite Plants in Medicago truncatula used for Nodulation Assays

16.0K Views.  Donald Danforth Plant Science Center. Si dimostra come le piante radice pelosa compositi possono essere utilizzati per studiare le interazioni pianta-Rhizobium e nodulazione nelle difficili da trasformare la specie Medicago truncatula.

Video: 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.

Saggi Neurocircuit per Sequestri in mutanti di Epilessia 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 ...

Ricerca

Biologia

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.

Analisi di Schwann-astrociti Interazioni utilizzo In Vitro Saggi

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.

Ibridazione in situ per la localizzazione precisa delle Trascrizioni nelle piante

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

Coupled Assays for Monitoring Protein Refolding in Saccharomyces cerevisiae

Proteostasis, defined as the combined processes of protein folding/biogenesis, refolding/repair, and degradation, is a delicate cellular balance that must be maintained to avoid deleterious consequences 1. External or internal factors that disrupt this balance can lead to protein aggregation, toxicity and cell death. In humans this is a major contributing factor to the symptoms associated with neurodegenerative disorders such as Huntington's, Parkinson's, and Alzheimer's diseases 10. It is therefore essential that the proteins involved in maintenance of proteostasis be identified in order to develop treatments for these debilitating diseases. This article describes techniques for monitoring in vivo protein folding at near-real time resolution using the model protein firefly luciferase fused to green fluorescent protein (FFL-GFP). FFL-GFP is a unique model chimeric protein as the FFL moiety is extremely sensitive to stress-induced misfolding and aggregation, which inactivates the enzyme 12. Luciferase activity is monitored using an enzymatic assay, and the GFP moiety provides a method of visualizing soluble or aggregated FFL using automated microscopy. These coupled methods incorporate two parallel and technically independent approaches to analyze both refolding and functional reactivation of an enzyme after stress. Activity recovery can be directly correlated with kinetics of disaggregation and re-solubilization to better understand how protein quality control factors such as protein chaperones collaborate to perform these functions. In addition, gene deletions or mutations can be used to test contributions of specific proteins or protein subunits to this process. In this article we examine the contributions of the protein disaggregase Hsp104 13, known to partner with the Hsp40/70/nucleotide exchange factor (NEF) refolding system 5, to protein refolding to validate this approach.

Coupled Assays for Monitoring Protein Refolding in Saccharomyces cerevisiae

This article describes the use of a firefly luciferase-GFP fusion protein to investigate in vivo protein folding in Saccharomyces cerevisiae. Using this reagent, refolding of a model heat-denatured protein can be monitored simultaneously by fluorescence microscopy and an enzymatic assay to probe the roles of proteostasis network components in protein quality control.

Saggi accoppiati per il monitoraggio delle proteine ​​in ripiegamento<em&gt; Saccharomyces cerevisiae</em

Proteostasis, defined as the combined processes of protein folding/biogenesis, refolding/repair, and degradation, is a delicate cellular balance that must ...

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.

I saggi di redditività per le cellule in coltura

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.

Efficiente generazione indotta umane pluripotenti cellule staminali da cellule somatiche umane con 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-Mass spettrometria di imaging per le inchieste sui metaboliti in<em&gt; Medicago truncatula</em&gt; Root noduli

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.

Misurazione spaziale e temporale Ca<sup&gt; 2 +</sup&gt; Segnali in Arabidopsis Piante

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.

Calibrazione e utilizzo delle prestazioni del microcoil MRM dimostrato sulle radici della trogola Medicago a 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.