Ce travail a été financé par une subvention de la National Science Foundation (numéro de subvention MCB-0447750).

<ol>
	<li>Clark, K. A., Krysan, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protocol:+An+improved+high-throughput+method+for+generating+tissue+samples+in+96-well+format+for+plant+genotyping+(Ice-Cap+2.0).">Protocol: An improved high-throughput method for generating tissue samples in 96-well format for plant genotyping (Ice-Cap 2.0).</a> Plant Methods. 3, 8-8 (2007).</li><li>Krysan, P. I. c. e. -. c. a. p. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+high-throughput+method+for+capturing+plant+tissue+samples+for+genotype+analysis.">A high-throughput method for capturing plant tissue samples for genotype analysis.</a> Plant Physiol. 135, 1162-1169 (2004).</li><li>Jander, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Arabidopsis+map-based+cloning+in+the+post-genome+era.">Arabidopsis map-based cloning in the post-genome era.</a> Plant Physiol. 129, 440-450 (2002).</li><li>Liljegren, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SHATTERPROOF+MADS-box+genes+control+seed+dispersal+in+Arabidopsis.">SHATTERPROOF MADS-box genes control seed dispersal in Arabidopsis.</a> Nature. 404, 766-770 (2000).</li><li>Buechel, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+A-type+ARABIDOPSIS+RESPONSE+REGULATORS+in+meristem+maintenance+and+regeneration.">Role of A-type ARABIDOPSIS RESPONSE REGULATORS in meristem maintenance and regeneration.</a> Eur. J. Cell. Biol. 89, 279-284 (2010).</li><li>Krysan, P. J., Jester, P. J., Gottwald, J. R., Sussman, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+Arabidopsis+mitogen-activated+protein+kinase+kinase+kinase+gene+family+encodes+essential+positive+regulators+of+cytokinesis.">An Arabidopsis mitogen-activated protein kinase kinase kinase gene family encodes essential positive regulators of cytokinesis.</a> Plant Cell. 14, 1109-1120 (2002).</li><li>Bush, S. M., Krysan, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=iTILLING:+a+personalized+approach+to+the+identification+of+induced+mutations+in+Arabidopsis.">iTILLING: a personalized approach to the identification of induced mutations in Arabidopsis.</a> Plant Physiol. 154, 25-35 (2010).</li><li>Comai, L., Henikoff, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TILLING:+practical+single-nucleotide+mutation+discovery.">TILLING: practical single-nucleotide mutation discovery.</a> Plant J. 45, 684-694 (2006).</li><li>Henikoff, S., Till, B. J., Comai, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TILLING.+Traditional+mutagenesis+meets+functional+genomics.">TILLING. Traditional mutagenesis meets functional genomics.</a> Plant Physiol. 135, 630-636 (2004).</li><li>McCallum, C. M., Comai, L., Greene, E. A., Henikoff, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeting+induced+local+lesions+IN+genomes+(TILLING)+for+plant+functional+genomics.">Targeting induced local lesions IN genomes (TILLING) for plant functional genomics.</a> Plant Physiol. 123, 439-442 (2000).</li></ol>

Pas de conflits d&#39;intérêt déclarés.

La méthode calotte présentée ici permet à un chercheur pour recueillir des échantillons de tissus provenant de centaines, voire des milliers, des plantes individuelles en une seule journée et efficace d&#39;extraire l&#39;ADN génomique à partir de ces échantillons pour une utilisation dans les réactions de génotypage. En donnant un scientifique de la capacité de déterminer le génotype des milliers de plants dans un court laps de temps, la méthode la calotte glaciaire a le potentiel pour faciliter un certain nombre d&#39;expériences génétiques qui autrement ne seraient pas pratiques à réaliser. Plusieurs exemples sont fournis ci-dessous. <ol><li> Cartographie génétique. Beaux échelle de cartographie génétique peut être accéléré si le chercheur est en mesure d&#39;identifier les événements de recombinaison dans un intervalle précis le long du chromosome 3. Si cet intervalle implique une très courte distance génétique, puis les événements de recombinaison sera relativement rare dans la population de cartographie de ségrégation. Pour cette raison, il peut être nécessaire à des milliers d&#39;écran de plantes individuelles pour trouver le nouveau désiréCombinant les individus. Ce processus pourrait être grandement accéléré par l&#39;utilisation de la calotte glaciaire. Même à l&#39;ère du génome entier de re-séquençage, il est encore nécessaire de démontrer que directement une mutation donnée est directement responsable d&#39;un phénotype d&#39;intérêt. Dans ces cas, il sera nécessaire de séparer la mutation d&#39;intérêt au détriment d&#39;autres mutations étroitement liées, afin de prouver une mutation candidat est effectivement responsable. Dans ces situations, la calotte glaciaire pourrait fournir une méthode efficace pour briser lien entre une paire de mutations étroitement liées. </li><li> Multi-mutants. Il est devenu commun pour les chercheurs qui étudient Arabidopsis pour combiner des allèles mutants de plusieurs loci distincts en un seul individu, afin de surmonter la redondance fonctionnelle 4-6. À titre d&#39;exemple, considérons un cas où l&#39;on souhaite combiner des allèles mutants de quatre loci différents dans une seule plante par croisement génétique. Pour ce projet, il sera nécessaire de générer uneParticulier N qui est hétérozygote à ces quatre loci dans le cadre du régime de passage. Lorsque cette graine quadruple hétérozygote ensembles, l&#39;homozygote quadruple est prévu d&#39;être présent à un taux de un sur 256 dans la population résultant de la descendance. Préalable de cette personne rare par les méthodes traditionnelles serait très laborieux. Par comparaison, en utilisant la calotte glaciaire pour ce projet permettrait au scientifique une stratégie efficace pour identifier les homozygotes rares quadrupler en une seule génération. </li><li> iTILLING. Notre laboratoire a récemment décrit une nouvelle approche pour le dépistage de la présence de mutations induites dans les gènes d&#39;intérêt dans 7 Arabidopsis. Cette méthode est une variation sur une technique bien établie appelée TILLING qui est utilisé pour trouver des mutations dans des populations commandé des individus traités avec un mutagene chimique 8-10. Nous avons nommé notre méthode iTILLING parce qu&#39;il s&#39;agit d&#39;une version personnalisée de TILLING qui permet une indchercheur ividual pour accomplir quelque chose qui a traditionnellement exigé une grande équipe de recherche à compléter. Ice-Cap fait partie intégrante de la méthode iTILLING 7, tel que décrit ci-dessous. </li></ol> L&#39;idée derrière iTILLING est de produire une population éphémère mutant, qui est en contraste avec les populations durables mutant créé pour les projets traditionnels TILLING. Pour iTILLING, les graines d&#39;une population en vrac M2 sont semées en 50 100 blocs Ice Cap, et l&#39;ADN est extrait en utilisant la méthode des calottes glaciaires. Plants d&#39;Arabidopsis sont ensuite placés à 4 ° C, alors que le dépistage de mutation est réalisée en utilisant l&#39;ADN calotte glaciaire preps. Plants de tomates sont conservés à 12 ° C. Une fois les plantules présentant des mutations désirées ont été identifiés, ils sont récupérés à partir des plaques de glace-Cap et transféré au sol. iTILLING n&#39;est pas destiné à fournir une population durable mutantes pour une communauté de recherche entier à l&#39;écran. Au lieu de cela, iTILLING fournit une stratégie par laquelleun scientifique peut filtrer une population mutante personnalisées pour des mutations dans une poignée de gènes rapidement, efficacement et à un coût relativement faible. Nous avons trouvé calotte glaciaire d&#39;être une méthode efficace pour la croissance et le génotypage d&#39;Arabidopsis, la tomate et du riz. Nous prévoyons que d&#39;autres espèces de plantes devraient également se prêter à calotte glaciaire. Une limite à la variété des espèces qui peuvent être logés par Ice-Cap est la taille des graines. Seules les plantes avec des graines assez petit pour tenir dans le puits d&#39;une plaque de 96 puits sera compatible avec la calotte glaciaire dans sa configuration actuelle.

<table border="1"><tbody><tr><td> Nom du réactif </td><td> Société </td><td> Numéro de catalogue </td><td> Commentaires (optionnel) </td></tr><tr><td> GenoGrinder </td><td> Spex / CertiPrep, Metuchen, NJ </td><td> 2000 Geno / Grinder </td><td> Machine pour secouer Plaques racine pour l&#39;extraction d&#39;ADN </td></tr><tr><td> ABgene Thermo-Scelleur </td><td> ABgene Unis </td><td> AB-0384 </td><td> Machine manuelle thermoscellage utilisée à des plaques de Racine avant de désorganisation tissulaire </td></tr><tr><td> Centrifugeuse équipée des supports de microplaques balancer </td><td> Beckman Coulter </td><td> Allegra 25R </td><td> Toute centrifugeuse avec des supports de microplaques balancer serait approprié </td></tr><tr><td> MicroFill microplaques Distributeur </td><td> BioTek www.biotek.com </td><td> AF1000A </td><td> Automated distributeur de liquide pour microplaques remplissant </td></tr><tr><td> Bialetti marque17x11 pouces Fiche commerciaux Cookie Grand </td><td> Target magasin </td><td> www.bialetti.com </td><td> Cette feuille de biscuit spécifique a la profondeur idéale pour la calotte glaciaire </td></tr><tr><td> Custom Made rack métallique </td><td> Quincaillerie </td><td></td><td> Voir le texte pour la description complète </td></tr><tr><td> 33 litres boîte de rangement en plastique </td><td> Target magasin </td><td> Sterilite Marque </td><td> Pour la calotte glaciaire Fontaine. Boîte de rangement en plastique assez grand pour contenir la plaque à biscuits 17x11 pouces </td></tr><tr><td> 30 gallons par heure Pompe de fontaine </td><td> Sunterra </td><td> 104506 </td><td> Petite pompe submersible fontaine disponible dans les magasins de fournitures de jardin </td></tr><tr><td> Tuyau pour pompe de fontaine </td><td> Toute entreprise de fournitures de laboratoire </td><td></td><td> Tuyau compatible avec pompe de fontaine </td></tr><tr><td> Pince plastique </td><td> Quincaillerie </td><td></td><td> Pince à tenir le tuyau à biscuitsFiche </td></tr><tr><td> Effacer les couvercles en plastique de Falcon Microtest fond plat de 96 puits des plaques de polystyrène </td><td> BD Falcon www.bdbiosciences.com </td><td> 351172 </td><td> Effacer les couvercles en plastique pour couvrir les plaques de glace semis-Cap </td></tr><tr><td> 3M Micropore ruban chirurgical </td><td> Fisher Scientific </td><td> 19-027-761 </td><td></td></tr><tr><td> 3/32-inch boules de diamètre en acier inoxydable </td><td> Technologies de Hartford, Rocky Hill, CT </td><td> 034 à 006-1K </td><td> Utilisé pour perturber les tissus des racines après la récolte </td></tr><tr><td> 3 pouces bandeaux élastique </td><td> Épicerie locale </td><td></td><td> Utilisé pour sécuriser Plate Racine à la plaque de semis </td></tr><tr><td> 4 brochettes en bois mm de diamètre </td><td> Épicerie locale </td><td></td><td> Ces brochettes sont généralement utilisés pour préparer les brochettes </td></tr><tr><td> Fisher Scientific Nunc marque de 1 ml plaques filtre sans fritte </td><td> Fisher Scientific </td><td> 278012 </td><td> Plaques de semis de 96 puits avec des trous dans le fond. </td></tr><tr><td> Fisherbrand 96-Eh bien Tall-cheminée Plaque PCR </td><td> Fisher Scientific </td><td> 14-230-242 </td><td> Plaques 96 puits à la racine </td></tr><tr><td> Agar - culture cellulaire testée </td><td> Sigma-Aldrich </td><td> A1296 </td><td></td></tr><tr><td> 4-Morpholinoethanesulfonic acide (MES) </td><td> Sigma-Aldrich </td><td> M2933 </td><td></td></tr><tr><td> Murashige et Skoog (MS) mélange de sel basale </td><td> Sigma-Aldrich </td><td> M-5524 </td><td></td></tr><tr><td> La chaleur Easy Peel étanchéité feuille </td><td> ABgene Unis </td><td> AB-0745 </td><td> Film d&#39;étanchéité pour l&#39;étanchéité thermique Plaques Racine avant de désorganisation tissulaire </td></tr><tr><td> Marque Canard bande extra large Emballage, 3,00 &quot;x 54,6 m </td><td> OfficeMax www.officemax.com </td><td> Réf. 21242139 </td><td> Ruban adhésifpour microplaques d&#39;étanchéité contenant des extraits d&#39;ADN pour le stockage </td></tr><tr><td> Rouleau d&#39;étanchéité </td><td> Bio-Rad </td><td> MSR-0001 </td><td> Hand held outil de rouler pour le pressage de bandes d&#39;étanchéité adhésif sur microplaques </td></tr><tr><td> 96 puits à bloc thermique du métal </td><td> Cole-Parmer </td><td> 36400-66 </td><td> Utilisé pour geler les plaques pour la récolte des racines du tissu </td></tr><tr><td> Papier-filtre Whatman </td><td> Whatman international </td><td> 1002 090 </td><td> Utilisé pour les graines de la surface de stérilisation </td></tr><tr><td> Pipette Pasteur jetable </td><td> Fisher Scientific </td><td> 13-678-6A </td><td> 5 ¾ &quot;jetables pipette Pasteur utilisée pour le transfert de semences à l&#39;assiette des semis </td></tr><tr><td> Pyrex plat allant au four (20 cm x 30 cm) </td><td> Target magasin </td><td></td><td> Utilisé pour construire une glace sèche / éthanol bain. </td></tr></tbody></table>

ice-cap: a method for growing arabidopsis and tomato plants in 96-well plates for high-throughput genotyping

Il est devenu courant pour les scientifiques des plantes pour développer des projets qui nécessitent le génotypage d&#39;un grand nombre de plantes. La première étape de tout projet de génotypage est de collecter un échantillon de tissu de chaque plante. L&#39;approche traditionnelle de cette tâche est de plantes échantillon de un en un temps. Si l&#39;on veut à des centaines ou des milliers de génotypes particuliers, cependant, l&#39;utilisation de ce résultat stratégie dans un goulot d&#39;étranglement important dans le pipeline de génotypage. La méthode la calotte glaciaire que nous décrivons ici fournit une solution haut-débit à ce défi en permettant à un chercheur de collecter des tissus de plusieurs milliers de plants dans un seul jour 1,2. Ce niveau de débit est rendue possible par le fait que le tissu est récolté à partir de plantes de 96 en un temps, plutôt que d&#39;une moins-un-temps. La méthode la calotte glaciaire offre une plate-forme intégrée pour effectuer la croissance des semis, la récolte des tissus, et l&#39;extraction d&#39;ADN. La base de la calotte glaciaire est la croissance de seedliNGS dans une paire empilée de plaques à 96 puits. Les puits de la plaque supérieure contiennent les bouchons de gélose de milieu de croissance sur lequel plants individuels germer. Les racines poussent vers le bas à travers les médias d&#39;agar, sortie de la plaque supérieure par un trou, et de passer dans une eau contenant la plaque inférieure. Pour la récolte de tissus pour l&#39;extraction d&#39;ADN, de l&#39;eau dans la plaque inférieure contenant les tissus des racines est rapidement congelé alors que les semis dans la plaque supérieure rester à température ambiante. La plaque supérieure est ensuite décollé de la plaque inférieure, ce qui donne une plaque avec 96 échantillons de tissu racinaire dans la glace et une plaque avec 96 plants viables. La technique est appelée &quot;Ice-Cap», car il utilise de la glace pour capturer le tissu racinaire. La plaque 96 puits contenant les plantules peuvent ensuite enveloppés dans du papier et transférés à basse température. Ce processus suspend une nouvelle croissance des plants, mais n&#39;affecte pas leur viabilité. Une fois l&#39;analyse du génotype a été achevé, les jeunes plants avec le génotype désiré peut être transférée de la p 96 puitsfin à la terre pour la multiplication ultérieure. Nous avons démontré l&#39;utilité de la méthode de la calotte glaciaire à l&#39;aide d&#39;Arabidopsis thaliana, la tomate, et les semis de riz. Nous prévoyons que la méthode doit également être applicable à d&#39;autres espèces de plantes à graines assez petit pour tenir dans les puits de plaques à 96 puits.

Watch this Scientific Journal Video about Ice-Cap: A Method for Growing Arabidopsis and Tomato Plants in 96-well Plates for High-Throughput Genotyping at JoVE.com

Ice-Cap: A Method for Growing Arabidopsis and Tomato Plants in 96-well Plates for High-Throughput Genotyping | Biology | Video

Ice-Cap: A Method for Growing Arabidopsis and Tomato Plants in 96-well Plates for High-Throughput Genotyping

La méthode calotte permet de cultiver des plantes en plaques à 96 puits et non destructive tissu racinaire de récolte de chaque plant. L&#39;ADN extrait à partir de ce tissu racinaire peut être utilisé pour des réactions de génotypage. Nous avons constaté que la calotte glaciaire fonctionne bien pour les<em&gt; Arabidopsis thaliana</em&gt;, La tomate, les plants de riz.

Ice-Cap: un procédé de croissance Arabidopsis Et plants de tomates dans des plaques à 96 puits pour génotypage à haut débit

It is becoming common for plant scientists to develop projects that require the genotyping of large numbers of plants. The first step in any genotyping ...

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Ice-Cap: A Method for Growing Arabidopsis and Tomato Plants in 96-well Plates for High-Throughput Genotyping

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22.7K vues.  University of Wisconsin-Madison. La méthode calotte permet de cultiver des plantes en plaques à 96 puits et non destructive tissu racinaire de récolte de chaque plant. L'ADN extrait à partir de ce tissu racinaire peut être utilisé pour des réactions de génotypage. Nous avons constaté que la calotte glaciaire fonctionne bien pour les Arabidopsis thaliana, La tomate, les plants de riz.

Vidéo: Ice-Cap: A Method for Growing Arabidopsis and Tomato Plants in 96-well Plates for High-Throughput Genotyping

Pyrosequencing: A Simple Method for Accurate Genotyping

Pharmacogenetic research benefits first-hand from the abundance of information provided by the completion of the Human Genome Project. With such a tremendous amount of data available comes an explosion of genotyping methods. Pyrosequencing(R) is one of the most thorough yet simple methods to date used to analyze polymorphisms. It also has the ability to identify tri-allelic, indels, short-repeat polymorphisms, along with determining allele percentages for methylation or pooled sample assessment. In addition, there is a standardized control sequence that provides internal quality control. This method has led to rapid and efficient single-nucleotide polymorphism evaluation including many clinically relevant polymorphisms. The technique and methodology of Pyrosequencing is explained.


Pyrosequencing(R) is one of the most thorough yet simple methods to date used to analyze polymorphisms. This method has led to rapid and efficient single-nucleotide polymorphism evaluation including many clinically relevant polymorphisms. The technique and methodology of Pyrosequencing is explained.

Pyroséquençage: une méthode simple pour le génotypage précis

Pharmacogenetic research benefits first-hand from the abundance of information provided by the completion of the Human Genome Project. With such a ...

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A High-Throughput Method For Zebrafish Sperm Cryopreservation and In Vitro Fertilization

This is a method for zebrafish sperm cryopreservation that is an adaptation of the Harvey method (Harvey et al., 1982).  We have introduced two changes to the original protocol that both streamline the procedure and increase sample uniformity.  First, we normalize all sperm volumes using freezing media that does not contain the cryoprotectant. Second, cryopreserved sperm are stored in cryovials instead of capillary tubes.  The rates of sperm freezing and thawing (&delta;&deg;C/time) are probably the two most critical variables to control in this procedure.  For this reason, do not substitute different tubes for those specified.  Working in teams of 2 it is possible to freeze the sperm of 100 males per team in ~2 hrs.  Sperm cryopreserved using this protocol has an average of 25% fertility (measured as the number of viable embryos generated in an in vitro fertilization divided by the total number of eggs fertilized) and this percent fertility is stable over many years.

A High-Throughput Method For Zebrafish Sperm Cryopreservation and In Vitro Fertilization

This is a high-throughput sperm cryopreservation protocol for zebrafish. Sperm cryopreserved using this protocol has an average of 25% fertility in subsequent vitro fertilization and is stable over many years.

Une méthode à haut débit pour la cryoconservation du sperme de poisson zèbre et de In Vitro La fertilisation

This is a method for zebrafish sperm cryopreservation that is an adaptation of the Harvey method (Harvey et al., 1982).  We have introduced two changes to ...

A Rapid High-throughput Method for Mapping Ribonucleoproteins (RNPs) on Human pre-mRNA

Sequencing RNAs that co-immunoprecipitate (co-IP) with RNA binding proteins has increased our understanding of splicing by demonstrating that binding location often influences function of a splicing factor.  However, as with any sampling strategy the chance of identifying an RNA bound to a splicing factor is proportional to its cellular abundance.  We have developed a novel in vitro approach for surveying binding specificity on otherwise transient pre-mRNA. This approach utilizes a specifically designed oligonucleotide pool that tiles across introns, exons, splice junctions, or other pre-mRNA.  The pool is subjected to some kind of molecular selection. Here, we demonstrate the method by separating the oligonucleotide into a bound and unbound fraction and utilize a two color array strategy to record the enrichment of each oligonucleotide in the bound fraction. The array data generates high-resolution maps with the ability to identify sequence-specific and structural determinates of ribonucleoprotein (RNP) binding on pre-mRNA.  A unique advantage to this method is its ability to avoid the sampling bias towards mRNA associated with current IP and SELEX techniques, as the pool is specifically designed and synthesized from pre-mRNA sequence. The flexibility of the oligonucleotide pool is another advantage since the experimenter chooses which regions to study and tile across, tailoring the pool to their individual needs.  Using this technique, one can assay the effects of polymorphisms or mutations on binding on a large scale or clone the library into a functional splicing reporter and identify oligonucleotides that are enriched in the included fraction.  This novel in vitro high-resolution mapping scheme provides a unique way to study RNP interactions with transient pre-mRNA species, whose low abundance makes them difficult to study with current in vivo techniques.  



A Rapid High-throughput Method for Mapping Ribonucleoproteins RNPs on Human pre-mRNA

Due to the transient nature of pre-mRNA, it can be difficult to isolate and study in vivo. Here, we present a novel in vitro approach to investigate RNA-protein interactions using a synthetic oligo pool that tiles across selected regions of pre-mRNA.

Un rapide à haut débit Méthode de cartographie ribonucléoprotéines (RNP) sur l&#39;homme pré-ARNm

Sequencing RNAs that co-immunoprecipitate (co-IP) with RNA binding proteins has increased our understanding of splicing by demonstrating that binding ...

Measuring Caenorhabditis elegans Life Span in 96 Well Microtiter Plates

Lifespan is a biological process regulated by several genetic pathways. One strategy to investigate the biology of aging is to study animals that harbor mutations in components of age-regulatory pathways. If these mutations perturb the function of the age-regulatory pathway and therefore alter the lifespan of the entire organism, they provide important mechanistic insights1-3.
Another strategy to investigate the regulation of lifespan is to use small molecules to perturb age-regulatory pathways. To date, a number of molecules are known to extend lifespan in various model organisms and are used as tools to study the biology of aging4-16. The number of molecules identified thus far is small compared to the genetic "toolset" that is available to study the biology of aging.
Caenorhabditis elegans is one of the principle models used to study aging because of its excellent genetics and short lifespan of three weeks. More recently, C.elegans has emerged as a model organism for phenotype based drug screens5,7,16-20 because of its small size and its ability to grow in microtiter plates.
Here we present an assay to measure C.elegans lifespan in 96 well microtiter plates. The assay was developed and successfully used to screen large libraries for molecules that extend C.elegans lifespan7. The reliability of the assay was evaluated in multiple tests: first, by measuring the lifespan of wild type animals grown at different temperatures; second, by measuring the lifespan of mutants with altered lifespans; third, by measuring changes in lifespan in response to different concentrations of the antidepressant Mirtazepine. Mirtazepine has previously been shown to extend lifespan in C.elegans7. The results of these tests show that the assay is able to replicate previous findings from other assays and is quantitative. The microtiter format also makes this lifespan assay compatible with automated liquid handling systems and allows integration into automated platforms.

Measuring Caenorhabditis elegans Life Span in 96 Well Microtiter Plates

In this protocol we present a method to measure Caenorhabditis elegans lifespan in 96 well microtiter plates.

Mesure Caenorhabditis elegans</emDurée de vie> à 96 plaques de microtitration bien

Lifespan is a biological process regulated by several genetic pathways. One strategy to investigate the biology of aging is to study animals that harbor ...

A High-throughput Method for Measurement of Glomerular Filtration Rate in Conscious Mice

The measurement of glomerular filtration rate (GFR) is the gold standard in kidney function assessment. Currently, investigators determine GFR by measuring the level of the endogenous biomarker creatinine or exogenously applied radioactive labeled inulin (3H or 14C). Creatinine has the substantial drawback that proximal tubular secretion accounts for ~50% of total renal creatinine excretion and therefore creatinine is not a reliable GFR marker. Depending on the experiment performed, inulin clearance can be determined by an intravenous single bolus injection or continuous infusion (intravenous or osmotic minipump). Both approaches require the collection of plasma or plasma and urine, respectively. Other drawbacks of radioactive labeled inulin include usage of isotopes, time consuming surgical preparation of the animals, and the requirement of a terminal experiment. Here we describe a method which uses a single bolus injection of fluorescein isothiocyanate-(FITC) labeled inulin and the measurement of its fluorescence in 1-2 &mu;l of diluted plasma. By applying a two-compartment model, with 8 blood collections per mouse, it is possible to measure GFR in up to 24 mice per day using a special work-flow protocol. This method only requires brief isoflurane anesthesia with all the blood samples being collected in a non-restrained and awake mouse. Another advantage is that it is possible to follow mice over a period of several months and treatments (i.e. doing paired experiments with dietary changes or drug applications). We hope that this technique of measuring GFR is useful to other investigators studying mouse kidney function and will replace less accurate methods of estimating kidney function, such as plasma creatinine and blood urea nitrogen.

Measurement of glomerular filtration rate (GFR) is the gold standard for kidney function assessment. Here we describe a high-throughput method which allows the determination of GFR in conscious mice by using a single bolus injection, determination of fluorescein isothiocyanate (FITC)-inulin in plasma and calculation of GFR by a two-phase exponential decay model.

Une méthode à haut débit pour la mesure de débit de filtration glomérulaire chez des souris conscientes

The measurement of  glomerular filtration rate (GFR) is the gold standard in kidney function  assessment. Currently, investigators determine GFR by ...

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+ ...

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 ...

Measuring Oxidative Stress Resistance of Caenorhabditis elegans in 96-well Microtiter Plates

Oxidative stress, which is the result of an imbalance between production and detoxification of reactive oxygen species, is a major contributor to chronic human disorders, including cardiovascular and neurodegenerative diseases, diabetes, aging, and cancer. Therefore, it is important to study oxidative stress not only in cell systems but also using whole organisms. C. elegans is an attractive model organism to study the genetics of oxidative stress signal transduction pathways, which are highly evolutionarily conserved.
Here, we provide a protocol to measure oxidative stress resistance in C. elegans in liquid. Briefly, ROS-inducing reagents such as paraquat (PQ) and H2O2 are dissolved in M9 buffer, and solutions are aliquoted in the wells of a 96 well microtiter plate. Synchronized L4/young adult C. elegans animals are transferred to the wells (5-8 animals/well) and survival is measured every hour until most worms are dead. When performing an oxidative stress resistance assay using a low concentration of stressors in plates, aging might influence the behavior of animals upon oxidative stress, which could lead to an incorrect interpretation of the data. However, in the assay described herein, this problem is unlikely to occur since only L4/young adult animals are being used. Moreover, this protocol is inexpensive and results are obtained in one day, which renders this technique attractive for genetic screens. Overall, this will help to understand oxidative stress signal transduction pathways, which could be translated into better characterization of oxidative stress-associated human disorders.

Measuring Oxidative Stress Resistance of Caenorhabditis elegans in 96-well Microtiter Plates

C. elegans is an attractive model organism to study signal transduction pathways involved in oxidative stress resistance. Here we provide a protocol to measure oxidative stress resistance of C. elegans animals in liquid phase, using several oxidizing agents in 96 well plates.

Mesurer le stress oxydatif Résistance<em&gt; Caenorhabditis elegans</em&gt; En 96 plaques de microtitration bien

Oxidative stress, which is the result of an imbalance between production and detoxification of reactive oxygen species, is a major contributor to chronic ...

qPCRTag Analysis - A High Throughput, Real Time PCR Assay for Sc2.0 Genotyping

The Synthetic Yeast Genome Project (Sc2.0) aims to build 16 designer yeast chromosomes and combine them into a single yeast cell. To date one synthetic chromosome, synIII1, and one synthetic chromosome arm, synIXR2, have been constructed and their in vivo function validated in the absence of the corresponding wild type chromosomes. An important design feature of Sc2.0 chromosomes is the introduction of PCRTags, which are short, re-coded sequences within open reading frames (ORFs) that enable differentiation of synthetic chromosomes from their wild type counterparts. PCRTag primers anneal selectively to either synthetic or wild type chromosomes and the presence/absence of each type of DNA can be tested using a simple PCR assay. The standard readout of the PCRTag assay is to assess presence/absence of amplicons by agarose gel electrophoresis. However, with an average PCRTag amplicon density of one per 1.5 kb and a genome size of ~12 Mb, the completed Sc2.0 genome will encode roughly 8,000 PCRTags. To improve throughput, we have developed a real time PCR-based detection assay for PCRTag genotyping that we call qPCRTag analysis. The workflow specifies 500 nl reactions in a 1,536 multiwell plate, allowing us to test up to 768 PCRTags with both synthetic and wild type primer pairs in a single experiment.

Designer chromosomes of the Synthetic Yeast Genome project, Sc2.0, can be distinguished from their native counterparts using a PCR-based genotyping assay called PCRTagging, which has a presence/absence endpoint. Here we describe a high-throughput real time PCR detection method for PCRTag genotyping.

Analyse qPCRTag - un débit élevé, PCR temps réel pour Sc2.0 Génotypage

The Synthetic Yeast Genome Project (Sc2.0) aims to build 16 designer yeast chromosomes and combine them into a single yeast cell. To date one synthetic ...

High-throughput CRISPR Vector Construction and Characterization of DNA Modifications by Generation of Tomato Hairy Roots

Targeted DNA mutations generated by vectors with clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 technology have proven useful for functional genomics studies. While most cloning strategies are simple to perform, they generally use multiple steps and can require several days to generate the ultimate constructs. The method presented here is based on DNA assembly and can produce fully functional CRISPR vectors in a single cloning reaction. Vector construction can also be pooled, further increasing the efficiency and utility of the process. A modification of the method is used to create CRISPR vectors with multiple gene targets. CRISPR vectors are then transformed into tomato hairy roots to generate transgenic materials with targeted DNA modifications. Hairy roots are a useful system for testing vector functionality as they are technically simple to generate and amenable to large-scale production. The methods presented here will have wide application as they can be used to generate a variety of CRISPR vectors and be used in a wide range of plant species.

Using DNA assembly, multiple CRISPR vectors can be constructed in parallel in a single cloning reaction, making the construction of large numbers of CRISPR vectors a simple task. Tomato hairy roots are an excellent model system to validate CRISPR vectors and generate mutant materials.

Haut-débit CRISPR Vector Construction et caractérisation de l&#39;ADN Modifications par génération de tomate Poilu Roots

Targeted DNA mutations generated by vectors with clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 technology have proven useful for ...