Diese Arbeit wurde durch ein Stipendium der National Science Foundation (Grant-Nummer MCB-0447750) gefördert.

<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>

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Die Ice-Cap hier vorgestellte Methode ermöglicht ein Wissenschaftler an Gewebeproben von Hunderten oder sogar Tausenden von einzelnen Pflanzen an einem einzigen Tag zu sammeln und effizient zu extrahieren genomischer DNA aus den Proben für den Einsatz in Genotypisierung Reaktionen. Durch die Aufgabe von einzelnen Wissenschaftler die Möglichkeit, Tausende von Setzlingen in einem kurzen Zeitraum Genotyp hat der Ice-Cap-Methode das Potenzial, eine Reihe von genetischen Experimenten, die sonst nicht sinnvoll sein, führen Sie erleichtern würde. Einige Beispiele sind unten angegeben. <ol><li> Genetic Mapping. Fine-Skala genetische Kartierung kann beschleunigt werden, wenn der Forscher in der Lage ist, um eine Rekombination Ereignisse innerhalb eines bestimmten Intervalls entlang der Chromosom 3 identifiziert werden. Wenn dieses Intervall mit einem sehr kurzen genetischen Distanz, dann die Rekombination Veranstaltungen werden relativ selten in der Trennung Mapping Bevölkerung. Aus diesem Grund kann es notwendig sein, um Bildschirm Tausende von einzelnen Pflanzen, um die gewünschte wieder findenrekombinante Individuen. Dieser Prozess könnte wesentlich durch den Einsatz von Ice-Cap beschleunigt werden. Auch im Zeitalter der Genom-weite Re-Sequenzierung, ist es noch notwendig, um direkt zu demonstrieren, dass eine bestimmte Mutation direkt verantwortlich für einen Phänotyp von Interesse ist. In diesen Fällen wird es notwendig sein, die interessierende Mutation weg zu trennen von anderen eng miteinander verknüpft Mutationen um zu beweisen, ein Kandidat Mutation tatsächlich ursächlich. In diesen Situationen würde Ice-Cap bieten eine effiziente Methode für das Brechen Verknüpfung zwischen einem Paar eng miteinander verbunden Mutationen. </li><li> Multi-Mutanten. Es hat sich für die Forscher studieren Arabidopsis, um mutierte Allele von mehreren verschiedenen Loci in einer einzigen Person zu kombinieren, um funktionelle Redundanz 4-6 überwinden gemeinsam. Als Beispiel betrachten wir einen Fall, wo man wünscht, Mutanten-Allele aus vier verschiedenen Loci kombinieren in einer einzigen Pflanze durch genetische Kreuzung. Für dieses Projekt wird es notwendig sein, einen zu generierenn Person, heterozygot ist an diesen vier loci als Teil der Kreuzung Schema. Wenn diese Vierfach-heterozygote Sets Saatgut ist die vierfache homozygote voraussichtlich mit einer Rate von einem von 256 in der daraus resultierenden Population von Nachkommen präsentieren. Screening für diesen seltenen Einzelfällen mit herkömmlichen Methoden wäre ziemlich arbeitsintensiv. Im Vergleich dazu würde mit Ice-Cap für dieses Projekt der Wissenschaftler mit einer effizienten Strategie zur Identifizierung der seltenen Vierfach-Homozygoten in einer einzigen Generation bieten. </li><li> iTILLING. Unser Labor kürzlich über einen neuen Ansatz zur Screening auf das Vorhandensein von induzierten Mutationen in Genen von Interesse in Arabidopsis 7. Diese Methode ist eine Variante einer etablierten Technik namens TILLING, dass wird verwendet, um Mutationen in geordneten Populationen von Individuen mit einem chemischen Mutagen 8-10 behandelt zu finden. Wir haben unsere Methode iTILLING genannt, weil es eine individualisierte Version des TILLING, dass ein ind erlaubt istividual Wissenschaftler zu erreichen etwas, das traditionell ein großes Forscherteam um komplette verlangt hat. Ice-Cap ist ein integraler Bestandteil der iTILLING Methode 7, wie unten beschrieben. </li></ol> Die Idee hinter iTILLING ist eine ephemere mutierten Bevölkerung, die im Gegensatz zu den langlebigen Mutanten Bevölkerung für traditionelle TILLING Projekte erstellt steht zu produzieren. Für iTILLING werden die Samen aus einer bulked M2 Bevölkerung in 50 bis 100 Ice Cap Blöcken gesät, und die DNA extrahiert wird mit dem Ice-Cap-Verfahren. Arabidopsis-Keimlinge sind dann bei 4 ° C, während Mutations-Screening erfolgt mit dem Ice-Cap DNA preps. Tomatenjungpflanzen sind bei 12 ° C gelagert. Wenn Sämlinge tragen gewünschten Mutationen identifiziert worden sind, werden sie aus dem Ice-Cap Platten wiederhergestellt und an Boden. iTILLING ist nicht beabsichtigt, eine langfristige mutierten Bevölkerung für eine gesamte Forschung auf dem Bildschirm sorgen. Stattdessen bietet iTILLING eine Strategie, durch dieein Wissenschaftler kann eine benutzerdefinierte mutierten Bevölkerung auf Mutationen in einer Handvoll von Genen schnell Bildschirm, effizient und auf einem relativ niedrigen Kosten. Wir haben Ice-Cap gefunden, um eine effektive Methode für den Anbau und die Genotypisierung Arabidopsis, Tomaten und Reis. Wir erwarten, dass andere Arten von Pflanzen sollte auch zugänglich sein Ice-Cap. Eine Einschränkung auf die Vielfalt der Arten, die von Ice-Cap untergebracht werden kann, ist die Größe der Samen. Nur Pflanzen mit Samen klein genug, um in die Vertiefungen einer 96-Well-Platte fit ist kompatibel mit Ice-Cap in seiner jetzigen Konfiguration.

<table border="1"><tbody><tr><td> Name des Reagenzes </td><td> Firma </td><td> Katalog-Nummer </td><td> Kommentare (optional) </td></tr><tr><td> GenoGrinder </td><td> Spex / CertiPrep, Metuchen, NJ </td><td> 2000 Geno / Grinder </td><td> Maschinen zum Schütteln Root-Platten für die DNA-Extraktion </td></tr><tr><td> ABgene Thermo-Sealer </td><td> ABgene USA </td><td> AB-0384 </td><td> Manuelle Heißsiegelmaschine verwendet werden, um Root-Platten vor Gewebeaufschluss </td></tr><tr><td> Zentrifuge ausgestattet mit schwingenden Mikrotiterplattenträger </td><td> Beckman Coulter </td><td> Allegra 25R </td><td> Jede Zentrifuge mit schwingenden Microplatte Träger geeignet wären </td></tr><tr><td> MICROFILL Microplate Dispenser </td><td> BioTek www.biotek.com </td><td> AF1000A </td><td> Automatisierte Flüssigkeitsspender zum Befüllen Mikrotiterplatten </td></tr><tr><td> Bialetti Marke17x11 Zoll Handelsregister großen Backblech </td><td> Ziel Kaufhaus </td><td> www.bialetti.com </td><td> Diese spezifische Backblech hat die ideale Tiefe für Ice-Cap </td></tr><tr><td> Custom Made Metallgestell </td><td> Eisenwarengeschäft </td><td></td><td> Siehe Text für vollständige Beschreibung </td></tr><tr><td> 33 Liter Kunststoff-Aufbewahrungsbox </td><td> Ziel Kaufhaus </td><td> Sterilite Marke </td><td> Für Ice-Cap-Brunnen. Kunststoff-Aufbewahrungsbox groß genug, um die 17x11 Zoll Backblech halten </td></tr><tr><td> 30 Gallonen pro Stunde Wasserspielpumpen </td><td> Sunterra </td><td> 104506 </td><td> Kleine Tauchpumpe Brunnen Pumpe unter Garten-Versorgung-Läden </td></tr><tr><td> Schlauch für Brunnen-Pumpe </td><td> Jedes Labor EVU </td><td></td><td> Hose mit Brunnen Pumpe </td></tr><tr><td> Plastic Clamp </td><td> Eisenwarengeschäft </td><td></td><td> Clamp-Schlauch zu halten CookieBlatt </td></tr><tr><td> Klare Kunststoffdeckel von Falcon Microtest flachem Boden 96-well Platten aus Polystyrol </td><td> BD Falcon www.bdbiosciences.com </td><td> 351172 </td><td> Klare Kunststoff Abdeckungen für Ice-Cap Sämling Plates </td></tr><tr><td> 3M Micropore chirurgischen Band </td><td> Fisher Scientific </td><td> 19-027-761 </td><td></td></tr><tr><td> 3/32-inch Durchmesser Edelstahlkugeln </td><td> Hartford Technologies, Rocky Hill, CT </td><td> 034 bis 006-1K </td><td> Wird verwendet, um Wurzelgewebe nach der Ernte zerstören </td></tr><tr><td> 3-Zoll-Elastic Haarbänder </td><td> Lokale Lebensmittelgeschäft </td><td></td><td> Wird verwendet, um Root-Platte zu Platte zu sichern Sämling </td></tr><tr><td> 4 mm Durchmesser Holzspieße </td><td> Lokale Lebensmittelgeschäft </td><td></td><td> Diese Spieße werden in der Regel verwendet, um kabobs vorbereiten </td></tr><tr><td> Fisher Scientific Nunc Marke 1-mL Filterplatten ohne Fritte </td><td> Fisher Scientific </td><td> 278012 </td><td> 96-well Platten Sämling mit Löchern im Boden. </td></tr><tr><td> Fisherbrand 96-Well Tall-Chimney PCR Plate </td><td> Fisher Scientific </td><td> 14-230-242 </td><td> 96-well Platten Wurzel </td></tr><tr><td> Agar - Zellkultur getestet </td><td> Sigma-Aldrich </td><td> A1296 </td><td></td></tr><tr><td> 4-Morpholinethansulfonsäure (MES) </td><td> Sigma-Aldrich </td><td> M2933 </td><td></td></tr><tr><td> Murashige und Skoog (MS) basalen Salzmischung </td><td> Sigma-Aldrich </td><td> M-5524 </td><td></td></tr><tr><td> Easy Peel Wärme Dichtfolie </td><td> ABgene USA </td><td> AB-0745 </td><td> Heißsiegelfolie zur Abdichtung Root-Platten vor Gewebeaufschluss </td></tr><tr><td> Ente Marke Extra Wide Tape, 3,00 &quot;x 54,6 m </td><td> OfficeMax www.officemax.com </td><td> Artikel-Nr. 21242139 </td><td> Klebebandzum Abdichten von Mikroplatten mit DNA-Extrakten für die Lagerung </td></tr><tr><td> Siegelwalze </td><td> Bio-Rad </td><td> MSR-0001 </td><td> Hand gehalten Rollwerkzeug zum Pressen Dichtband auf Mikrotiterplatten </td></tr><tr><td> 96-Loch-Metall Thermoblock </td><td> Cole-Parmer </td><td> 36400-66 </td><td> Wird verwendet, um die Root-Platten für die Gewebe-Ernte eingefroren </td></tr><tr><td> Whatman Filterpapier </td><td> Whatman International </td><td> 1002 090 </td><td> Wird für die Oberfläche Sterilisation Samen </td></tr><tr><td> Einweg-Pasteur Pipette </td><td> Fisher Scientific </td><td> 13 bis 678-6A </td><td> 5 ¾ &quot;Einweg-Pasteur-Pipette für die Übertragung von Samen Sämling Platte verwendet </td></tr><tr><td> Pyrex Backform (20 cm x 30 cm) </td><td> Ziel Kaufhaus </td><td></td><td> Wird verwendet, um einem Trockeneis / Ethanol-Bad zu bauen. </td></tr></tbody></table>

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

Es wird immer für den Anlagenbau Wissenschaftler gemeinsam Projekte, die die Genotypisierung einer großen Zahl von Anlagen erfordern entwickeln. Der erste Schritt in eine Genotypisierung Projektes ist es, eine Gewebeprobe aus einzelnen Pflanzen zu sammeln. Der traditionelle Ansatz, um diese Aufgabe ist es, Probe Pflanzen one-at-a-time. Will man Genotyp Hunderte oder Tausende von Einzelpersonen, aber mit dieser Strategie führt zu einer deutlichen Engpass bei der Genotypisierung Pipeline. Die Ice-Cap-Methode, die wir hier beschreiben, ist ein High-Throughput-Lösung für diese Herausforderung, indem ein Wissenschaftler, um Gewebe aus mehreren tausend Sämlinge an einem einzigen Tag 1,2 sammeln. Dieses Maß an Durchsatz wird durch die Tatsache, dass Gewebe aus Pflanzen ist 96-at-a-time geerntet, sondern als ein-at-a-time. Die Ice-Cap-Verfahren stellt eine integrierte Plattform für die Durchführung Sämlingwachstum-, Gewebe-Ernte, und die DNA-Extraktion. Die Grundlage für die Ice-Cap ist das Wachstum der seedlings in einem gestapelten Paar von 96-Well-Platten. Die Brunnen der oberen Platte enthalten Stecker von Agar Nährmedien, auf denen einzelne Sämlinge keimen. Die Wurzeln wachsen nach unten durch den Agar-Medien, beenden Sie das obere Platte durch ein Loch, und gelangen in eine untere Platte mit Wasser. Zur Ernte Gewebe zur DNA-Extraktion, das Wasser in der unteren Platte, die Wurzel Gewebe schnell eingefroren, solange die Keimlinge in der oberen Platte bei Raumtemperatur bleiben. Die obere Platte wird dann von der unteren Platte, geschält Nachgeben einer Platte mit 96 root Gewebeproben in Eis und eine Platte mit 96 lebensfähige Sämlinge eingefroren. Die Technik trägt den Namen &quot;Ice-Cap&quot;, weil es Eis verwendet, um die Wurzel Gewebe zu erfassen. Der 96-Well-Platte mit den Setzlingen kann dann in Folie verpackt und auf niedriger Temperatur. Dieser Prozess setzt ein weiteres Wachstum der Sämlinge, aber keinen Einfluss auf ihre Lebensfähigkeit. Nach Analyse des Genotyps abgeschlossen ist, können Sämlinge mit den gewünschten Genotyp aus dem 96-Loch-p übertragen werdenspät, um Boden für die weitere Vermehrung. Wir haben den Nutzen des Ice-Cap-Verfahren mit Arabidopsis thaliana, Tomaten und Reis Sämlinge unter Beweis gestellt. Wir erwarten, dass die Methode sollte auch für andere Arten von Pflanzen mit Samen klein genug, um in die Vertiefungen von 96-Well-Platten passen.

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

Die Ice-Cap-Verfahren erlaubt es, Pflanzen in 96-Well-Platten und zerstörungsfrei Ernte Wurzelgewebe von jedem Sämling wachsen. DNA von dieser Wurzel Gewebe extrahiert werden können für die Genotypisierung Reaktionen eingesetzt werden. Wir haben festgestellt, dass Ice-Cap gut für<em&gt; Arabidopsis thaliana</em&gt;, Tomaten und Reis Sämlinge.

Ice-Cap: Ein Verfahren zur Züchtung von Arabidopsis Und Tomatenpflanzen in 96-well-Platten für High-Throughput-Genotypisierung

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 Aufrufe.  University of Wisconsin-Madison. Die Ice-Cap-Verfahren erlaubt es, Pflanzen in 96-Well-Platten und zerstörungsfrei Ernte Wurzelgewebe von jedem Sämling wachsen. DNA von dieser Wurzel Gewebe extrahiert werden können für die Genotypisierung Reaktionen eingesetzt werden. Wir haben festgestellt, dass Ice-Cap gut für Arabidopsis thaliana, Tomaten und Reis Sämlinge.

Serie: 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.

Pyrosequencing: Eine einfache Methode für genaue Genotypisierung

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

Forschung

Biologie

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.

Ein High-Throughput-Methode für Zebrabärblinge Sperma Kryokonservierung und In Vitro 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.

Eine schnelle High-Throughput-Verfahren zur Abbildung Ribonukleoproteine ​​(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 ...

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.

Messung Caenorhabditis elegans Life Span in 96 Well Mikrotiterplatten

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.

Ein High-Throughput-Verfahren zur Messung der glomerulären Filtrationsrate in Conscious Mäuse

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.

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

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

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.

Hochdurchsatz-Screening für Chemische Modulatoren der Post-transkriptionell regulierten Genen

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.

Mess Oxidativer Stress Widerstandsrat<em&gt; Caenorhabditis elegans</em&gt; In 96-Well-Mikrotiterplatten

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.

qPCRTag Analyse - ein hoher Durchsatz, Echtzeit-PCR-Assay für 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 ...

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.

High-throughput CRISPR Vector Konstruktion und Charakterisierung von DNA-Modifikationen durch Erzeugung von Tomaten Hairy Roots

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