Name: high throughput microinjections of sea urchin zygotes
Uploaded: 2014-01-21T21:00:00
Description: Watch this Scientific Journal Video about High Throughput Microinjections of Sea Urchin Zygotes at JoVE.com

Nous remercions Santiago Suarez pour la lecture critique du manuscrit et Betty Cowgill à l&#39;aide de la photographie. Nous remercions également les relecteurs anonymes pour leurs commentaires critiques. Ce travail est soutenu par l&#39;Université du Delaware Fonds de recherche.

Une. Préparation de Sulfate de protamine (PS) Plats couché <ol><li> Préparer une solution à 1% de sulfate de protamine (PS) en ajoutant 0,5 g de PS à 50 ml d&#39;eau désionisée, de l&#39;eau distillée (FD 2 O) dans un tube conique de 50 ml. Bien agiter à grande vitesse sur un agitateur de banc à la température ambiante pendant 1-2 heures pour assurer la dissolution complète de PS. Cette solution peut être conservé à 4 ° C pendant au moins 3 mois (assurez-vous de dissoudre complètement préc...

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A., Gasiorowski, J. Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nonviral+gene+delivery.">Nonviral gene delivery.</a> Cold Spring Harb. Protoc. , (2011).</li><li>Hiramoto, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+division+without+mitotic+apparatus+in+sea+urchin+eggs.">Cell division without mitotic apparatus in sea urchin eggs.</a> Exp. Cell Res. 11, 630-636 (1956).</li><li>Wilson, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Methods in Cell Biology. 25, (1982).</li><li>Gross, J. M., McClay, D. 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G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autonomy+in+specification+of+primordial+germ+cells+and+their+passive+translocation+in+the+sea+urchin.">Autonomy in specification of primordial germ cells and their passive translocation in the sea urchin.</a> Development. 139, 3786-3794 (2012).</li><li>Duboc, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nodal+and+BMP2/4+pattern+the+mesoderm+and+endoderm+during+development+of+the+sea+urchin+embryo.">Nodal and BMP2/4 pattern the mesoderm and endoderm during development of the sea urchin embryo.</a> Development. 137, 223-235 (2010).</li><li>Ernst, S. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Offerings+from+an+Urchin.">Offerings from an Urchin.</a> Dev. Biol. 358, 285-294 (2011).</li><li>McClay, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evolutionary+crossroads+in+developmental+biology:+sea+urchins.">Evolutionary crossroads in developmental biology: sea urchins.</a> Development. 138, 2639-2648 (2011).</li><li>Sodergren, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Research+article+-+The+genome+of+the+sea+urchin+Strongylocentrotus+purpuratus.">Research article - The genome of the sea urchin Strongylocentrotus purpuratus.</a> Science. 314, 941-952 (2006).</li><li>Roux, M. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+functional+genomic+and+proteomic+perspective+of+sea+urchin+calcium+signaling+and+egg+activation.">A functional genomic and proteomic perspective of sea urchin calcium signaling and egg activation.</a> Dev. Biol. 300, 416-433 (2006).</li><li>Turner, P. R., Sheetz, M. P., Jaffe, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fertilization+increases+the+polyphosphoinositide+content+of+sea+urchin+eggs.">Fertilization increases the polyphosphoinositide content of sea urchin eggs.</a> Nature. 310, 414-415 (1984).</li><li>Voronina, E., Marzluff, W. F., Wessel, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cyclin+B+synthesis+is+required+for+sea+urchin+oocyte+maturation.">Cyclin B synthesis is required for sea urchin oocyte maturation.</a> Dev. Biol. 256, 258-275 (2003).</li><li>Wong, J. L., Wessel, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extracellular+matrix+modifications+at+fertilization:+regulation+of+dityrosine+crosslinking+by+transamidation.">Extracellular matrix modifications at fertilization: regulation of dityrosine crosslinking by transamidation.</a> Development. 136, 1835-1847 (2009).</li><li>Shuster, C. B., Burgess, D. 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Acad. Sci. U.S.A. 105, 5955-5962 (2008).</li><li>Peter, I. S., Davidson, E. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+gene+regulatory+network+controlling+the+embryonic+specification+of+endoderm.">A gene regulatory network controlling the embryonic specification of endoderm.</a> Nature. 474, (2011).</li><li>Nam, J. M., Dong, P., Tarpine, R., Istrail, S., Davidson, E. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+cis-regulatory+genomics+for+systems+biology.">Functional cis-regulatory genomics for systems biology.</a> Proc. Natl. Acad. Sci. U.S.A. 107, 3930-3935 (2010).</li><li>Showman, R. M., Foerder, C. 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La micro-injection est une technique puissante pour délivrer divers traitements tels que l&#39;ADN, l&#39;ARNm, des protéines recombinantes, une perte de fonction et de gain de fonction de réactifs, les colorants et leurs combinaisons dans des œufs, des embryons et des cellules de divers organismes 7.1. Toutefois, plusieurs considérations doivent être gardées à l&#39;esprit lors de la conception d&#39;une expérience de micro-injection. Il est extrêmement important de pre...

L&#39;administration du traitement efficace et reproductible est l&#39;un des principaux défis méthodologiques pour les chercheurs. Plusieurs méthodes ont été mis en place pour fournir des solutions de traitement transitoire dans les oeufs, embryons et les cellules. Ces procédés comprennent l&#39;électroporation (basé sur une génération de pores transitoires de la membrane à l&#39;aide de courtes impulsions électriques) 1,2, la lipofection (livraison par la fusion de liposomes contenant au traitement avec la membrane) 1, 1 bombardement de microparticules (ADN est précipité sur le micron particules de taille métalliques qui sont ensuite utilisés pour pénétrer dans les cellules à grande vitesse), et la transduction (virus est utilisé en tant que véhicule de délivrance de transgènes). Pour le moment, la micro-injection est la seule approche qui détient l&#39;avantage de délivrer une solution avec 100% d&#39;efficacité avec des réactifs minimes. En outre, une solution d&#39;injection unique peut être composée d&#39;un mélange complexe de traitements. Cette technique a été utilisée avec succès pourment micro-injection des ovules et des embryons de nombreuses espèces tels que les oursins 3,4, poisson zèbre 5, 6 souris, grenouille 7, 8 et bovins ainsi que des cellules individuelles dans la culture de tissus 9. Blastomères simples injections à des stades de développement ultérieurs ont également été menées 10-12. Les méthodes actuelles de micro-injections sont basés sur la méthode injection sous pression qui a été initialement décrit par Hiramoto 10, mais de grands progrès ont été accomplis en vue de l&#39;optimisation de ce processus. D&#39;excellentes techniques de micro-injection ont été décrites ailleurs 11, et ici nous décrire l&#39;une des méthodes spécifiques qui sont actuellement utilisés pour micro-injection oursin (Strongylocentrotus purpuratus) des oeufs fécondés récemment. Depuis plus d&#39;un siècle, les oursins ont été un précieux modèle expérimental 15,16. Les oursins sont étroitement liées à l&#39;évolution, les chordés (nous y compris) et l&#39;analyse deleur génome a révélé qu&#39;ils contiennent toutes les grandes familles de gènes que l&#39;homme 17. Ils produisent un grand nombre de pays en synchrone embryons transparents qui peuvent être facilement manipulées. Utilisation de l&#39;oursin comme un organisme modèle, la communauté de l&#39;oursin a contribué à notre compréhension du processus de la fécondation 18-21, les processus biologiques cellulaires 22-24, et les réseaux de régulation génétique (GRNS) 25-28. Microinjection dans des zygotes d&#39;oursins nécessite plusieurs étapes. Tout d&#39;abord, les œufs doivent être immobilisées avant les injections (décrit ci-dessous). plats de microinjection sont revêtues avec de la protamine sulfate (PS), qui crée une surface chargée positivement à laquelle les oeufs chargés négativement peuvent adhérer 3. Les oeufs sont dejellied par incubation dans l&#39;eau de mer acide (pH 5,15) pendant 10 min, suivie par deux lavages dans l&#39;eau de mer naturelle ou de l&#39;eau de mer artificielle (pH 8,0). Les œufs sont soigneusement dejellied ramé en ligne droiteau milieu du plat revêtu PS en présence de 1 mM de 3-aminotriazole (3-AT), qui est nécessaire pour inhiber l&#39;activité de ovoperoxidase qui est sécrétée à partir des granules corticaux de l&#39;œuf à la suite de la fécondation 29. Cette étape est importante pour empêcher le durcissement de l&#39;enveloppe de la fécondation et de faciliter la pénétration de l&#39;aiguille de micro-injection. Comme alternative à 1 mM 3-AT, 10 mM d&#39;acide paraminobenzoic (PABA) peut être utilisé. La solution d&#39;injection est chargé dans une aiguille de micro-injection en utilisant spécialisé microloading pointe de pipette et monté sur un support fixé au micromanipulateur et de l&#39;unité de pression (figure 1). Chaque aiguille peut être utilisé pour micro-injection zygotes individuels dans de multiples expériences à des jours différents. La micro-injection peut être effectuée pendant 10-15 min jusqu&#39;à ce que les zygotes durcissent. Les zygotes sont ensuite lavées avec de l&#39;eau de mer et on cultive à 15 ° C. Lorsque les embryons atteignent l&#39;éclosion blastula stade, ils libèrent une enzyme qui digère l&#39;éclosion composantes du fertilization enveloppe 30 et leur permettent de se détacher naturellement de la PS plat revêtu. Si nécessaire, les embryons peuvent être doucement détachées de la boîte à l&#39;aide d&#39;une pipette de bouche ou une pipette Pasteur en soufflant doucement de l&#39;eau de mer sur les embryons. La méthode décrite permet la micro-injection efficace et fiable de 100-400 oeufs fécondés récemment sur un plat unique, fournissant une méthode à haut débit pour les analyses en aval.

<table border="0" cellpadding="0" cellspacing="0" style="width:793px;" width="794">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Glass pasteur pipettes</td>
			<td style="width:200px;">VWR</td>
			<td style="width:211px;">14673-043</td>
			<td style="width:167px;"> </td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Inverted microscope Axiovert 40C</td>
			<td style="width:200px;">Zeiss</td>
			<td style="width:211px;">4109431007990000</td>
			<td>Injection microscope</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Microloader tips</td>
			<td style="width:200px;">Eppendorf</td>
			<td style="width:211px;">5242 956.003</td>
			<td style="width:167px;">Load injection solution</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Nylon filter mesh 80 &#956;m</td>
			<td style="width:200px;">Amazon.com</td>
			<td style="width:211px;">03-80-37</td>
			<td style="width:167px;">Filter eggs to get rid of debris</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">P20 or P200 Aerosol Barrier Pipette Tips</td>
			<td style="width:200px;">Fisher Scientific</td>
			<td style="width:211px;">02707432 or 02707430</td>
			<td style="width:167px;">Part of a mouth pipette</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Parafilm</td>
			<td style="width:200px;">Fisher Scientific</td>
			<td style="width:211px;">13 374 12</td>
			<td style="width:167px;">Part of a mouth pipette</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Polyethylene tubing</td>
			<td style="width:200px;">Intramedic</td>
			<td style="width:211px;">PE-160</td>
			<td style="width:167px;">Part of a mouth pipette</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Protamine sulfate</td>
			<td style="width:200px;">MP Biomedicals, LLC</td>
			<td style="width:211px;">194729</td>
			<td style="width:167px;">Attach dejellied eggs to injection dishes</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Sea urchins S. purpuratus</td>
			<td>Pt. Loma Marine Invertebrate Lab</td>
			<td style="width:211px;">N/A</td>
			<td style="width:167px;"> </td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Sea water</td>
			<td style="width:200px;">any pet store</td>
			<td style="width:211px;">Instant Ocean</td>
			<td style="width:167px;"> </td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Sterile 60 mm x 15 mm Polystyrene Petri Dish</td>
			<td style="width:200px;">Fisher Scientific</td>
			<td style="width:211px;">0875713A</td>
			<td style="width:167px;">Injection dishes</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Three-Axis Coarse Positioning Micromanipulator MMN-1</td>
			<td style="width:200px;">Narishige</td>
			<td style="width:211px;">9124</td>
			<td>Manipulate injection needle</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:216px;">Three-Axis Joystick Type Oil Hydraulic Fine Micromanipulator MMO-202ND</td>
			<td style="width:200px;">Narishige</td>
			<td style="width:211px;">9212</td>
			<td>Manipulate injection needle</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Transfer pipettes</td>
			<td style="width:200px;">Fisher Scientific</td>
			<td style="width:211px;">13-711-9AM</td>
			<td style="width:167px;"> </td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Vertical needle puller </td>
			<td style="width:200px;">Narishige</td>
			<td style="width:211px;">PC-10</td>
			<td>Pull injection needles</td>
		</tr>
	</tbody>
</table>

high throughput microinjections of sea urchin zygotes

La micro-injection dans des cellules et des embryons est une technique couramment utilisée pour étudier un grand nombre de processus biologiques. Dans ce procédé, une petite quantité de solution de traitement est chargée dans une aiguille de micro-injection qui est utilisée pour injecter physiquement les cellules immobilisées ou des embryons individuels. Malgré la nécessité d&#39;une formation initiale pour effectuer cette procédure pour la livraison à haut débit, la micro-injection offre un maximum d&#39;efficacité et de livraison reproductible d&#39;une grande variété de solutions de traitement (y compris les mélanges complexes d&#39;échantillons) dans les cellules, les ovules ou d&#39;embryons. Applications aux micro-injections comprennent la livraison de constructions d&#39;ADN, ARNm, protéines recombinantes, gain de fonction, et la perte de réactifs de fonction. Colorant fluorescent ou colorimétrique est ajouté à la solution injectée pour permettre la visualisation instantanée de la livraison efficace, ainsi que d&#39;un outil pour la normalisation fiable de la quantité de la solution fournie. La méthode décrite permet la micro-injection de 100-400 oursin zygotes dans 10-15 min.

GFP et mCherry constructions rapporteurs ont été transcrits in vitro et micro-injecté dans les œufs nouvellement fécondés. Les embryons ont été incubées à 15 ° C pendant 24 heures (jusqu&#39;à ce que le stade blastula) et imagées par Zeiss Observateur Z1 microscope. Injection de constructions rapporteurs n&#39;a pas conduit à des défauts de développement (figure 6). Pour la quantification de signaux de fluorescence, l&#39;acquisition des images a été effectuée à un faible gr...

Watch this Scientific Journal Video about High Throughput Microinjections of Sea Urchin Zygotes at JoVE.com

High Throughput Microinjections of Sea Urchin Zygotes

La micro-injection est une technique couramment utilisée pour fournir des constructions d&#39;ADN, ARNm, des oligonucléotides morpholino antisens ou d&#39;autres traitements dans les œufs, les embryons et les cellules de diverses espèces.

À haut débit microinjections oursins zygotes

Microinjection into cells and embryos is a common technique that is used to study a wide range of biological processes. In this method a small amount of ...

high-throughput-microinjections-of-sea-urchin-zygotes

Research

JoVE Journal

Biology

Intravenous Microinjections of Zebrafish Larvae to Study Acute Kidney Injury

In this video article we describe a zebrafish model of AKI using gentamicin as the nephrotoxicant.  The technique consists of intravenous microinjections on 2 dpf zebrafish. This technique represents an efficient and rapid method to deliver soluble substances into the bloodstream of zebrafish larvae, allowing for the injection of 15-20 fish per hour. In addition to AKI studies, this microinjection technique can also be used for other types of experimental studies such as angiography.  We provide a detailed protocol of the technique from equipment required to visual measures of decreased kidney function. In addition, we also demonstrate the process of fixation, whole mount immunohistochemistry with a kidney tubule marker, plastic embedding and sectioning of the larval zebrafish. We demonstrate that zebrafish larvae injected with gentamicin show morphological features consistent with AKI: edema, loss of cell polarity in proximal tubular epithelial cells, and morphological disruption of the tubule.

We describe a technique of microinjecting the aminoglycoside, gentamicin, into 2 days post-fetilization (dpf) zebrafish larvae to induce acute kidney injury (AKI). We also describe a method for whole mount immunohistochemistry, plastic embedding and sectioning of zebrafish larvae to visualize the AKI mediated damage.

Microinjections intraveineuse de larves de poisson zèbre pour étudier lésions rénales aiguës

In this video article we describe a zebrafish model of AKI using gentamicin as the nephrotoxicant.  The technique consists of intravenous microinjections ...

Recherche

Biologie

High-throughput Yeast Plasmid Overexpression Screen

The budding yeast, Saccharomyces cerevisiae, is a powerful model system for defining fundamental mechanisms of many important cellular processes, including those with direct relevance to human disease. Because of its short generation time and well-characterized genome, a major experimental advantage of the yeast model system is the ability to perform genetic screens to identify genes and pathways that are involved in a given process. Over the last thirty years such genetic screens have been used to elucidate the cell cycle, secretory pathway, and many more highly conserved aspects of eukaryotic cell biology 1-5. In the last few years, several genomewide libraries of yeast strains and plasmids have been generated 6-10. These collections now allow for the systematic interrogation of gene function using gain- and loss-of-function approaches 11-16. Here we provide a detailed protocol for the use of a high-throughput yeast transformation protocol with a liquid handling robot to perform a plasmid overexpression screen, using an arrayed library of 5,500 yeast plasmids. We have been using these screens to identify genetic modifiers of toxicity associated with the accumulation of aggregation-prone human neurodegenerative disease proteins. The methods presented here are readily adaptable to the study of other cellular phenotypes of interest.

Here we describe a plasmid overexpression screen in Saccharomyces cerevisiae, using an arrayed plasmid library and a high-throughput yeast transformation protocol with a liquid handling robot.

Haut débit écran surexpression plasmide de levure

The budding yeast, Saccharomyces cerevisiae, is a powerful model system for defining fundamental mechanisms of many important cellular processes, ...

High-throughput Saccharification Assay for Lignocellulosic Materials

Polysaccharides that make up plant lignocellulosic biomass can be broken down to produce a range of sugars that subsequently can be 
used in establishing a biorefinery. These raw materials would constitute a new industrial platform, which is both sustainable and carbon neutral, to replace 
the current dependency on fossil fuel. The recalcitrance to deconstruction observed in lignocellulosic materials is produced by several intrinsic properties 
of plant cell walls. Crystalline cellulose is embedded in matrix polysaccharides such as xylans and arabinoxylans, and the whole structure is encased by the 
phenolic polymer lignin, that is also difficult to digest 1. In order to improve the digestibility of plant materials we need to discover the main bottlenecks 
for the saccharification of cell walls and also screen mutant and breeding populations to evaluate the variability in saccharification 2. These tasks require 
a high throughput approach and here we present an analytical platform that can perform saccharification analysis in a 96-well plate format. This platform has 
been developed to allow the screening of lignocellulose digestibility of large populations from varied plant species. We have scaled down the reaction volumes 
for gentle pretreatment, partial enzymatic hydrolysis and sugar determination, to allow large numbers to be assessed rapidly in an automated system.
This automated platform works with milligram amounts of biomass, performing ball milling under controlled conditions to reduce the plant 
materials to a standardised particle size in a reproducible manner. Once the samples are ground, the automated formatting robot dispenses specified and recorded 
amounts of material into the corresponding wells of 96 deep well plate (Figure 1). Normally, we dispense the same material into 4 wells to have 4 replicates for 
analysis. Once the plates are filled with the plant material in the desired layout, they are manually moved to a liquid handling station (Figure 2). In this 
station the samples are subjected to a mild pretreatment with either dilute acid or alkaline and incubated at temperatures of up to 90&deg;C. The pretreatment solution 
is subsequently removed and the samples are rinsed with buffer to return them to a suitable pH for hydrolysis. The samples are then incubated with an enzyme
mixture for a variable length of time at 50&deg;C. An aliquot is taken from the hydrolyzate and the reducing sugars are automatically determined by the MBTH 
colorimetric method.

A simple, rapid method for determining the saccharification potential of large numbers of plant biomass samples is described. The automated platform for this analysis involves the preparation of the plant biomass for analysis in 96 well plates and the subsequent performance of pretreatment, hydrolysis and quantification of the sugars released.

Assay saccharification haut débit pour les matériaux lignocellulosiques

Polysaccharides that make up plant lignocellulosic biomass can be broken down to produce a range of sugars that subsequently can be 
used in establishing ...

High-throughput Purification of Affinity-tagged Recombinant Proteins

X-ray crystallography is the method of choice for obtaining a detailed view of the structure of proteins. Such studies need to be complemented by further biochemical analyses to obtain detailed insights into structure/function relationships. Advances in oligonucleotide- and gene synthesis technology make large-scale mutagenesis strategies increasingly feasible, including the substitution of target residues by all 19 other amino acids. Gain- or loss-of-function phenotypes then allow systematic conclusions to be drawn, such as the contribution of particular residues to catalytic activity, protein stability and/or protein-protein interaction specificity.
In order to attribute the different phenotypes to the nature of the mutation - rather than to fluctuating experimental conditions - it is vital to purify and analyse the proteins in a controlled and reproducible manner. High-throughput strategies and the automation of manual protocols on robotic liquid-handling platforms have created opportunities to perform such complex molecular biological procedures with little human intervention and minimal error rates1-5.
Here, we present a general method for the purification of His-tagged recombinant proteins in a high-throughput manner. In a recent study, we applied this method to a detailed structure-function investigation of TFIIB, a component of the basal transcription machinery. TFIIB is indispensable for promoter-directed transcription in vitro and is essential for the recruitment of RNA polymerase into a preinitiation complex6-8. TFIIB contains a flexible linker domain that penetrates the active site cleft of RNA polymerase9-11. This linker domain confers two biochemically quantifiable activities on TFIIB, namely (i) the stimulation of the catalytic activity during the 'abortive' stage of transcript initiation, and (ii) an additional contribution to the specific recruitment of RNA polymerase into the preinitiation complex4,5,12 . We exploited the high-throughput purification method to generate single, double and triple substitution and deletions mutations within the TFIIB linker and to subsequently analyse them in functional assays for their stimulation effect on the catalytic activity of RNA polymerase4. Altogether, we generated, purified and analysed 381 mutants - a task which would have been time-consuming and laborious to perform manually. We produced and assayed the proteins in multiplicates which allowed us to appreciate any experimental variations and gave us a clear idea of the reproducibility of our results.
This method serves as a generic protocol for the purification of His-tagged proteins and has been successfully used to purify other recombinant proteins. It is currently optimised for the purification of 24 proteins but can be adapted to purify up to 96 proteins.

We describe a method for the affinity-tagged purification of recombinant proteins using liquid-handling robotics. This method is generally applicable to the small-scale purification of soluble His-tagged proteins in a high-throughput format.

À haut débit Affinity Purification de protéines recombinantes-étiquette

X-ray crystallography is the method of choice for obtaining a detailed view of the structure of proteins. Such studies need to be complemented by further ...

Flow Cytometry-based Purification of S. cerevisiae Zygotes

Zygotes are essential intermediates between haploid and diploid states in the life cycle of many organisms, including yeast (Figure 1) 1. S. cerevisiae zygotes result from the fusion of haploid cells of distinct mating type (MATa, MATalpha) and give rise to corresponding stable diploids that successively generate as many as 20 diploid progeny as a result of their strikingly asymmetric mitotic divisions 2. Zygote formation is orchestrated by a complex sequence of events: In this process, soluble mating factors bind to cognate receptors, triggering receptor-mediated signaling cascades that facilitate interruption of the cell cycle and culminate in cell-cell fusion. Zygotes may be considered a model for progenitor or stem cell function.
Although much has been learned about the formation of zygotes and although zygotes have been used to investigate cell-molecular questions of general significance, almost all studies have made use of mating mixtures in which zygotes are intermixed with a majority population of haploid cells 3-8. Many aspects of the biochemistry of zygote formation and the continuing life of the zygote therefore remain uninvestigated.
Reports of purification of yeast zygotes describe protocols based on their sedimentation properties 9; however, this sedimentation-based procedure did not yield nearly 90% purity in our hands. Moreover, it has the disadvantage that cells are exposed to hypertonic sorbitol. We therefore have developed a versatile purification procedure. For this purpose, pairs of haploid cells expressing red or green fluorescent proteins were co-incubated to allow zygote formation, harvested at various times, and the resulting zygotes were purified using a flow cytometry-based sorting protocol. This technique provides a convenient visual assessment of purity and maturation. The average purity of the fraction is approximately 90%. According to the timing of harvest, zygotes of varying degrees of maturity can be recovered. The purified samples provide a convenient point of departure for "-omic" studies, for recovery of initial progeny, and for systematic investigation of this progenitor cell.

Flow Cytometry-based Purification of S. cerevisiae Zygotes

To purify zygotes of S. cerevisiae, haploid cells of opposite mating type were engineered to express red or green fluorescent proteins, co-incubated to allow zygote formation, and fractionated using a flow cytometry-based protocol. The highly-enriched fraction enables subsequent "-omic" studies, recovery of initial progeny, and systematic investigation of zygote morphogenesis.

Cytométrie en flux à base de purification de<em&gt; S. cerevisiae</em&gt; Zygotes

Zygotes are essential intermediates between haploid and diploid states in the life cycle of many organisms, including yeast (Figure 1) 1. S. cerevisiae ...

High-throughput Titration of Luciferase-expressing Recombinant Viruses

Standard plaque assays to determine infectious viral titers can be time consuming, are not amenable to a high volume of samples, and cannot be done with viruses that do not form plaques. As an alternative to plaque assays, we have developed a high-throughput titration method that allows for the simultaneous titration of a high volume of samples in a single day. This approach involves infection of the samples with a Firefly luciferase tagged virus, transfer of the infected samples onto an appropriate permissive cell line, subsequent addition of luciferin, reading of plates in order to obtain luminescence readings, and finally the conversion from luminescence to viral titers. The assessment of cytotoxicity using a metabolic viability dye can be easily incorporated in the workflow in parallel and provide valuable information in the context of a drug screen. This technique provides a reliable, high-throughput method to determine viral titers as an alternative to a standard plaque assay.

This article presents a high-throughput luciferase expression-based method of titrating various RNA and DNA viruses using automated and manual liquid handlers.

Le titrage à haut débit de la luciférase-virus recombinants exprimant

Standard plaque assays to determine infectious viral titers can be time consuming, are not amenable to a high volume of samples, and cannot be done with ...

A Method for Microinjection of Patiria minata Zygotes

Echinoderms have long been a favorite model system for studies of reproduction and development, and more recently for the study of gene regulation and evolution of developmental processes. The sea star, Patiria miniata, is gaining prevalence as a model system for these types of studies which were previously performed almost exclusively in the sea urchins, Strongylocentrotus purpuratus and Lytechinus variegatus. An advantage of these model systems is the ease of producing modified embryos in which a particular gene is up or downregulated, labeling a group of cells, or introducing a reporter gene. A single microinjection method is capable of creating a wide variety of such modified embryos. Here, we present a method for obtaining gametes from P. miniata, producing zygotes, and introducing perturbing reagents via microinjection. Healthy morphant embryos are subsequently isolated for quantitative and qualitative studies of gene function. The availability of genome and transcriptome data for this organism has increased the types of studies that are performed and the ease of executing them.

A Method for Microinjection of Patiria minata Zygotes

Methods that produce morphant embryos are essential to study developmental mechanisms and gene regulatory networks. The sea star Patiria miniata is an emerging model system for these studies. Here we present a protocol for obtaining gametes, producing cultures of embryos, and rapid microinjection of zygotes from this species.

Une méthode pour micro-injection de<em&gt; Patiria minata</em&gt; zygotes

Echinoderms have long been a favorite model system for studies of reproduction and development, and more recently for the study of gene regulation and ...

Recovery of Adult Zebrafish Hearts for High-throughput Applications

Use of the zebrafish model system for studying development, regeneration, and disease is expanding toward use of adult hearts for cell dissociation and purification of RNA, DNA, and proteins. All of these applications demand the rapid recovery of significant numbers of zebrafish hearts to avoid gene regulatory, metabolic, and other changes that begin after death. Adult zebrafish hearts are also required for studying heart structure for a variety of mutants and for studying heart regeneration. However, the traditional zebrafish heart dissection is slow and difficult and requires specialized tools, making large-scale dissection of adult zebrafish hearts tedious. Traditional methods also harbor the risk of damaging the heart during the dissection. Here, we describe a method for dissection of adult zebrafish hearts that is fast, reproducible, and preserves heart architecture. Furthermore, this method does not require specialized tools, is painless for the zebrafish, can be performed on fresh or fixed specimens, and can be performed on zebrafish as young as one month old. The approach described expands the use of adult zebrafish for cardiovascular research.

Use of zebrafish for cardiovascular research is expanding towards research on adult hearts. For these applications, quick and simple isolation of cardiac tissues is key to avoid post-mortem changes and to obtain an adequate number of samples. Here, we describe a fast and reproducible method for dissecting adult zebrafish hearts.

Recouvrement des adultes coeurs de poisson zèbre à haut débit pour des applications

Use of the zebrafish model system for studying development, regeneration, and disease is expanding toward use of adult hearts for cell dissociation and ...

High-throughput Screening for Chemical Modulators of Post-transcriptionally Regulated Genes

Both transcriptional and post-transcriptional regulation have a profound impact on genes expression. However, commonly adopted cell-based screening assays focus on transcriptional regulation, being essentially aimed at the identification of promoter-targeting molecules. As a result, post-transcriptional mechanisms are largely uncovered by gene expression targeted drug development. Here we describe a cell-based assay aimed at investigating the role of the 3&#39; untranslated region (3&#8217; UTR) in the modulation of the fate of its mRNA, and at identifying compounds able to modify it. The assay is based on the use of a luciferase reporter construct containing the 3&#8217; UTR of a gene of interest stably integrated into a disease-relevant cell line. The protocol is divided into two parts, with the initial focus on the primary screening aimed at the identification of molecules affecting luciferase activity after 24 hr of treatment. The second part of the protocol describes the counter-screening necessary to discriminate compounds modulating luciferase activity specifically through the 3&#8217; UTR. In addition to the detailed protocol and representative results, we provide important considerations about the assay development and the validation of the hit(s) on the endogenous target. The described cell-based reporter gene assay will allow scientists to identify molecules modulating protein levels via post-transcriptional mechanisms dependent on a 3&#8217; UTR.

Here we describe a cell-based reporter gene assay as a valuable tool to screen chemical libraries for compounds modulating post-transcriptional control mechanisms exerted through 3&#8217; UTR.

Haut-débit criblage de modulateurs chimique des gènes post-transcriptionnelle réglementées

Both transcriptional and post-transcriptional regulation have a profound impact on genes expression. However, commonly adopted cell-based screening assays ...

Identification of Kinase-substrate Pairs Using High Throughput Screening

We have developed a screening platform to identify dedicated human protein kinases for phosphorylated substrates which can be used to elucidate novel signal transduction pathways. Our approach features the use of a library of purified GST-tagged human protein kinases and a recombinant protein substrate of interest. We have used this technology to identify MAP/microtubule affinity-regulating kinase 2 (MARK2) as the kinase for a glucose-regulated site on CREB-Regulated Transcriptional Coactivator 2 (CRTC2), a protein required for beta cell proliferation, as well as the Axl family of tyrosine kinases as regulators of cell metastasis by phosphorylation of the adaptor protein ELMO. We describe this technology and discuss how it can help to establish a comprehensive map of how cells respond to environmental stimuli.

Protein phosphorylation is a central feature of how cells interpret and respond to information in their extracellular milieu. Here, we present a high throughput screening protocol using kinases purified from mammalian cells to rapidly identify kinases that phosphorylate a substrate(s) of interest.

Identification de paires Kinase-substrat en utilisant criblage à haut débit


We have developed a screening platform to identify dedicated human protein kinases for phosphorylated substrates which can be used to elucidate novel ...