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

हम फोटोग्राफी में सहायता के लिए पांडुलिपि और बेटी Cowgill के महत्वपूर्ण पढ़ने के लिए सैंटियागो सुआरेज़ धन्यवाद. हम भी उनके महत्वपूर्ण प्रतिक्रिया के लिए अनाम समीक्षक धन्यवाद. इस काम के विश्वविद्यालय डेलावेयर रिसर्च फंड द्वारा समर्थित है.

1. Protamine सल्फेट की तैयारी (पी एस) व्यंजन लेपित <ol><li> एक 50 मिलीलीटर शंक्वाकार ट्यूब में विआयनीकृत, आसुत जल (DDH 2 हे) के 50 एमएल के निजी सचिव की 0.5 ग्राम जोड़कर protamine सल्फेट (पी एस) के 1% समाधान तैयार करें. पुनश्च का पूर...

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Microinjection डीएनए, mRNA, पुनः संयोजक प्रोटीन, समारोह के नुकसान और अंडे, भ्रूण, और विभिन्न जीवों 1-7 की कोशिकाओं में समारोह अभिकर्मकों, रंग और उनके संयोजन के लाभ के रूप में विभिन्न उपचार देने के लिए एक शक्तिशाली ...

कुशल और प्रतिलिपि प्रस्तुत करने योग्य उपचार वितरण शोधकर्ताओं के लिए मुख्य methodological चुनौतियों में से एक है. कई तरीकों क्षणिक अंडे, भ्रूण, और कोशिकाओं में उपचार के समाधान देने के लिए स्थापित किया गया है. इन तरीकों में 1,2, lipofection (झिल्ली के साथ उपचार युक्त liposomes के विलय के माध्यम से वितरण) 1, microparticle बमबारी 1 (डीएनए माइक्रोन पर उपजी है (कम बिजली दालों का उपयोग झिल्ली में एक पैदा क्षणिक pores के आधार पर) electroporation शामिल आकार के धातु तब उच्च वेग में कोशिकाओं घुसना करने के लिए इस्तेमाल कर रहे हैं कि कण), और पारगमन (वायरस transgenes की एक डिलीवरी वाहन के रूप में प्रयोग किया जाता है). फिलहाल, microinjection न्यूनतम अभिकर्मकों के साथ 100% दक्षता के साथ किसी भी समाधान पहुंचाने का लाभ रखती है कि केवल दृष्टिकोण है. इसके अलावा, एक इंजेक्शन समाधान उपचार की एक जटिल कॉकटेल से बना जा सकता है. इस तकनीक को सफल करने के लिए इस्तेमाल किया गया हैly ऐसे समुद्री अर्चिन 3,4, ज़ेबरा मछली 5, माउस 6, मेंढक 7, और मवेशियों 8 के साथ ही टिशू कल्चर 9 में एकल कक्षों के रूप में कई प्रजातियों से अंडे और भ्रूण microinject. बाद में विकास के चरणों में एकल blastomeres इंजेक्शन भी 10-12 आयोजित किया गया है. microinjections के मौजूदा तरीकों शुरू में Hiramoto 10 द्वारा बताया गया था कि दबाव इंजेक्शन पद्धति पर आधारित हैं, लेकिन, महान प्रगति इस प्रक्रिया का अनुकूलन की दिशा में किया गया है. बहुत बढ़िया microinjection तकनीक कहीं 11 में वर्णित किया गया है, और यहाँ हम वर्तमान में समुद्री साही (Strongylocentrotus purpuratus) नव निषेचित अंडे microinject के लिए प्रयोग किया जाता है कि विशिष्ट विधियों में से एक का वर्णन. एक सदी से भी अधिक के लिए, समुद्री अर्चिन एक मूल्यवान प्रयोगात्मक मॉडल 15,16 किया गया है. सागर अर्चिन evolutionarily बारीकी (हमारे सहित) Chordates और का विश्लेषण करने के लिए संबंधित हैंउनके जीनोम वे मानव के रूप में 17 के सभी प्रमुख जीन परिवारों होते हैं कि पता चला. वे synchronously आसानी से छेड़छाड़ की जा सकती है कि पारदर्शी भ्रूण के विकास की एक बड़ी संख्या का उत्पादन. एक मॉडल के रूप में जीव समुद्री साही का प्रयोग, समुद्री साही समुदाय हमारे निषेचन प्रक्रिया की समझ 18-21, सेल जैविक प्रक्रियाओं 22-24, और जीन विनियामक नेटवर्क (GRNs) 25-28 में योगदान दिया है. समुद्री साही युग्मनजों में microinjection कई चरणों की आवश्यकता है. सबसे पहले, अंडे प्रायर (नीचे वर्णित) इंजेक्शन के लिए स्थिर हो की जरूरत है. Microinjection व्यंजन जो नकारात्मक आरोप लगाया अंडे 3 पालन कर सकते हैं करने के लिए एक सकारात्मक आरोप लगाया सतह बनाता है जो protamine सल्फेट (पीएस), के साथ लेपित हैं. अंडे प्राकृतिक समुद्र के पानी या कृत्रिम समुद्र के पानी (8.0 पीएच) में दो washes के द्वारा पीछा किया, 10 मिनट के लिए अम्लीय पानी समुद्र में ऊष्मायन (पीएच 5.15) द्वारा dejellied रहे हैं. dejellied अंडे ध्यान से एक सीधी रेखा में rowed रहे हैंनिषेचन 29 का एक परिणाम के रूप में अंडे के cortical कणिकाओं से स्रावित होता है कि ovoperoxidase की गतिविधि को बाधित करने के लिए आवश्यक है जो 1 मिमी 3 aminotriazole (3) में, की उपस्थिति में पुनश्च लेपित पकवान के बीच में. यह कदम निषेचन लिफाफा का सख्त को रोकने के लिए और microinjection सुई प्रवेश की सुविधा के लिए महत्वपूर्ण है. 1 मिमी 3 में करने के लिए एक विकल्प के रूप में 10 मिमी paraminobenzoic एसिड (PABA) का उपयोग किया जा सकता है. इंजेक्शन समाधान विशेष microloading पिपेट टिप का उपयोग कर एक microinjection सुई में भरी हुई है और micromanipulator और दबाव यूनिट से जुड़े एक धारक (चित्रा 1) पर मुहिम शुरू की है. प्रत्येक सुई अलग दिनों पर कई प्रयोगों में व्यक्तिगत युग्मनजों microinject करने के लिए इस्तेमाल किया जा सकता है. युग्मनजों कठोर जब तक microinjection 10-15 मिनट के लिए किया जा सकता है. युग्मनजों फिर 15 डिग्री सेल्सियस पर समुद्र के पानी के साथ और सुसंस्कृत धो रहे हैं भ्रूण ब्लासटुला चरण सेने तक पहुँचते हैं, वे fer के घटकों हज़म कि अंडे सेने एंजाइम रिलीजtilization लिफाफा 30 और उन्हें स्वाभाविक रूप से पी एस लेपित पकवान से अलग करने के लिए अनुमति देते हैं. यदि आवश्यक हो, भ्रूण धीरे धीरे भ्रूण पर समुद्र का पानी बह द्वारा एक मुँह पिपेट या पाश्चर पिपेट का उपयोग पकवान से अलग किया जा सकता है. वर्णित विधि बहाव के विश्लेषण के लिए एक उच्च throughput विधि उपलब्ध कराने, एक ही पकवान पर 100-400 नव निषेचित अंडे के कुशल और विश्वसनीय microinjection सक्षम बनाता है.

<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

कोशिकाओं और भ्रूण में microinjection जैविक प्रक्रियाओं की एक विस्तृत श्रृंखला का अध्ययन करने के लिए प्रयोग किया जाता है कि एक आम तकनीक है. इस विधि में उपचार समाधान की एक छोटी राशि शारीरिक रूप से व्यक्ति immobilized कोशिकाओं या भ्रूण इंजेक्षन करने के लिए प्रयोग किया जाता है कि एक microinjection सुई में भरी हुई है. उच्च throughput वितरण के लिए इस प्रक्रिया का प्रदर्शन करने के लिए प्रारंभिक प्रशिक्षण के लिए आवश्यकता के बावजूद, microinjection अधिकतम क्षमता और कोशिकाओं, अंडे या भ्रूण में (नमूने के जटिल मिश्रण सहित) के इलाज के समाधान की एक विस्तृत विविधता की प्रतिलिपि प्रस्तुत करने योग्य वितरण प्रदान करता है. Microinjections के लिए आवेदन डीएनए निर्माणों, mRNAs, पुनः संयोजक प्रोटीन, समारोह के लाभ, और समारोह अभिकर्मकों के नुकसान का वितरण शामिल है. फ्लोरोसेंट या वर्णमिति डाई तत्काल कुशल प्रसव के दृश्य के रूप में अच्छी तरह के रूप में दिया समाधान की राशि की विश्वसनीय सामान्य बनाने के लिए एक उपकरण को सक्षम करने के लिए इंजेक्शन समाधान के लिए जोड़ा है. वर्णित विधि 100-400 समुद्री साही Z के microinjection सक्षम बनाता है10-15 मिनट के भीतर ygotes.

GFP और mCherry संवाददाता निर्माणों लिखित और नव निषेचित अंडे में microinjected विट्रो में थे. भ्रूण (ब्लासटुला चरण तक) 24 घंटे के लिए 15 डिग्री सेल्सियस पर incubated और जीस प्रेक्षक Z1 माइक्रोस्कोप का उपयोग imaged थे. संवाददाता constru...

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

High Throughput Microinjections of Sea Urchin Zygotes

Microinjection अंडे, भ्रूण, और विभिन्न प्रजातियों की कोशिकाओं में डीएनए निर्माणों, mRNAs, morpholino antisense oligonucleotides या अन्य उपचार देने के लिए इस्तेमाल एक आम तकनीक है.

समुद्री साही युग्मनजों के उच्च throughput microinjections

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.

Zebrafish तीव्र गुर्दे चोट अध्ययन लार्वा के अंतःशिरा Microinjections

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

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.

उच्च throughput खमीर प्लाज्मिड Overexpression स्क्रीन

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.

Lignocellulosic सामग्री के लिए उच्च throughput Saccharification परख

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.

आत्मीयता टैग पुनः संयोजक प्रोटीन के शोधन उच्च throughput

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.

फ्लो आधारित शोधन<em&gt; एस 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.

Luciferase व्यक्त संयोजक वायरस के उच्च throughput अनुमापन

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.

के microinjection के लिए एक विधि<em&gt; Patiria minata</em&gt; युग्मनजों

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.

उच्च throughput अनुप्रयोगों के लिए वयस्क zebrafish दिल की वसूली

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.

पोस्ट-transcriptionally विनियमित जीन का रासायनिक Modulators के लिए उच्च throughput स्क्रीनिंग

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.

उच्च Throughput स्क्रीनिंग का उपयोग किनेस-सब्सट्रेट जोड़े की पहचान


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