Name: associated chromosome trap for identifying long-range dna interactions
Uploaded: 2011-04-23T14:00:00
Duration: 14 min 49 s
Description: Watch this Scientific Journal Video about Associated Chromosome Trap for Identifying Long-range DNA Interactions at JoVE.com

हम Adelle Murell और वुल्फ Reik उनके 3C प्रोटोकॉल साझा करने के लिए बहुत बहुत धन्यवाद. इस काम के रक्षा और दिग्गजों मामलों के विभाग के अनुसंधान सेवा के विभाग द्वारा समर्थित किया गया था.

<ol>
	<li>Dekker, J., Rippe, K., Dekker, M., Kleckner, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Capturing+chromosome+conformation.">Capturing chromosome conformation.</a> Science. 295, 1306-1311 (2002).</li><li>Apostolou, E., Thanos, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Virus+Infection+Induces+NF-kappaB-dependent+interchromosomal+associations+mediating+monoallelic+IFN-beta+gene+expression.">Virus Infection Induces NF-kappaB-dependent interchromosomal associations mediating monoallelic IFN-beta gene expression.</a> Cell. 134, 85-96 (2008).</li><li>Duan, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+three-dimensional+model+of+the+yeast+genome.">A three-dimensional model of the yeast genome.</a> Nature. 465, 363-367 (2010).</li><li>Lomvardas, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interchromosomal+interactions+and+olfactory+receptor+choice.">Interchromosomal interactions and olfactory receptor choice.</a> Cell. 126, 403-413 (2006).</li><li>Murrell, A., Heeson, S., Reik, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interaction+between+differentially+methylated+regions+partitions+the+imprinted+genes+Igf2+and+H19+into+parent-specific+chromatin+loops.">Interaction between differentially methylated regions partitions the imprinted genes Igf2 and H19 into parent-specific chromatin loops.</a> Nat Genet. 36, 889-893 (2004).</li><li>Schoenfelder, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preferential+associations+between+co-regulated+genes+reveal+a+transcriptional+interactome+in+erythroid+cells.">Preferential associations between co-regulated genes reveal a transcriptional interactome in erythroid cells.</a> Nat Genet. 42, 53-61 (2010).</li><li>Spilianakis, C. G., Lalioti, M. D., Town, T., Lee, G. R., Flavell, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interchromosomal+associations+between+alternatively+expressed+loci.">Interchromosomal associations between alternatively expressed loci.</a> Nature. 435, 637-645 (2005).</li><li>Vakoc, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proximity+among+distant+regulatory+elements+at+the+beta-globin+locus+requires+GATA-1+and+FOG-1.">Proximity among distant regulatory elements at the beta-globin locus requires GATA-1 and FOG-1.</a> Mol Cell. 17, 453-462 (2005).</li><li>Xu, N., Tsai, C. L., Lee, J. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transient+homologous+chromosome+pairing+marks+the+onset+of+X+inactivation.">Transient homologous chromosome pairing marks the onset of X inactivation.</a> Science. 311, 1149-1152 (2006).</li><li>Lieberman-Aiden, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comprehensive+mapping+of+long-range+interactions+reveals+folding+principles+of+the+human+genome.">Comprehensive mapping of long-range interactions reveals folding principles of the human genome.</a> Science. 326, 289-293 (2009).</li><li>Berkum, N. L. v. a. n. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hi-C:+a+method+to+study+the+three-dimensional+architecture+of+genomes.">Hi-C: a method to study the three-dimensional architecture of genomes.</a> J Vis Exp. , (2010).</li><li>Ling, J. Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CTCF+mediates+interchromosomal+colocalization+between+Igf2/H19+and+Wsb1/Nf1.">CTCF mediates interchromosomal colocalization between Igf2/H19 and Wsb1/Nf1.</a> Science. 312, 269-272 (2006).</li><li>Dekker, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+three+'C's+of+chromosome+conformation+capture:+controls,+controls,+controls.">The three 'C's of chromosome conformation capture: controls, controls, controls.</a> Nat Methods. 3, 17-21 (2006).</li></ol>

ब्याज की कोई संघर्ष की घोषणा की.

डेकर एट अल. गुणसूत्र रचना कब्जा दृष्टिकोण (3C) विकसित करने के लिए दो जीनोमिक loci 1 के बीच बातचीत की आवृत्ति का पता लगाने, और 3C बड़े पैमाने पर इस्तेमाल किया गया है दो स्तनधारी 2 कोशिकाओं में ज्ञात डीएनए क्षेत्रों के बीच अंतर गुणसूत्र और अंतर - गुणसूत्र संघों की जांच -9. हालांकि नव विकसित हाय - सी पद्धति जीनोम चौड़ा डीएनए संघ के अध्ययन के लिए लागू किया जा सकता है, अधिनियम परख अभी भी ठिकाना विशिष्ट डीएनए 10-11 बातचीत के अध्ययन के लिए एक प्रभावी तकनीक है. हम इस दृष्टिकोण को संशोधित किया है कि सभ्य माउस और मानव कोशिकाओं (चित्रा 1) में एक ज्ञात डीएनए क्षेत्र के साथ जुड़े रहे हैं अज्ञात डीएनए क्षेत्रों की पहचान. हम जुड़े गुणसूत्र जाल परख (अधिनियम) इस पद्धति का नाम है, क्योंकि यह हमें एक विश्वसनीय और प्रतिलिपि प्रस्तुत करने योग्य विधि उपन्यास अज्ञात डीएनए भागीदार है कि एक ज्ञात लक्ष्य डीएनए 12 क्षेत्र के साथ सहयोगी की पहचान प्रदान की. उचित नियंत्रण के साथ एक सफल 3C परख अधिनियम 13 परख के उपन्यास पहलुओं को क्रियान्वित करने से पहले किया जाता है. कई संभव के रूप में जुड़े डीएनए क्षेत्रों के रूप में tofind क्रम में, यह पहली और दूसरी प्रतिबंध एंजाइमों के विभिन्न संयोजनों का उपयोग करने के लिए आवश्यक है. यह विशेष रूप से प्रतिबंध एंजाइमों कि CPG मेथिलिकरण के लिए असंवेदनशील पहली 3C ligation कदम प्रदर्शन कर रहे हैं का उपयोग करने के लिए महत्वपूर्ण है. प्रोटीन बंधन और डीएनए मेथिलिकरण भी प्रतिबंध एंजाइम पाचन दक्षता को प्रभावित कर सकते हैं और जुड़े कुछ प्रतिबंध एंजाइम digestions के लिए डीएनए क्षेत्रों के ligation की विफलता के लिए नेतृत्व कर सकते हैं. अंतर या अंतर - गुणसूत्र ligation की घटना प्रोटीन डीएनए पार से जोड़ने और जुड़े डीएनए क्षेत्रों में दोनों के उचित शारीरिक नक्शे पर निर्भर करता है. इस प्रकार, कुछ प्रारंभिक प्रयोगों अधिनियम परख में एक साध्य प्रभावी formaldehyde एकाग्रता और उपचार समय की स्थापना के लिए आवश्यक हैं. Formaldehyde (from1.5% से 2%) के अंतिम सांद्रता का एक सीमा 7,9 परख के 3C भाग के दौरान कई प्रयोगशालाओं में इस्तेमाल किया गया है. वैकल्पिक रूप से, linkers के रूप में इस्तेमाल किया oligonucleotides एकजुट दूसरी प्रतिबंध एंजाइम द्वारा काट अंत मैच बनाया जा सकता है है. हालांकि हमने पाया है कि पीसीआर के दूसरे दौर में पीसीआर और 20-25 चक्र के पहले दौर में 18-20 चक्र स्पष्ट बैंड प्रदान कर सकता है, यह आवश्यक है कि प्रत्येक प्रयोग (चित्रा 2) के लिए सबसे अच्छा पीसीआर स्थितियों स्थापित इंट्रा अणु. 5&#39;-अंत और 3&#39;अंत एक कतरा डीएनए पीसीआर में हो सकता है की पूरक एडाप्टर अनुक्रम के बीच annealing एडाप्टर विशिष्ट डीएनए अणु के साथ annealing प्राइमर रोकता है, और पहले कई चक्रों में बहुत कम प्रवर्धन दक्षता में हुई. बाद लक्ष्य डीएनए चक्र के लिए परिलक्षित किया गया था, अपनी राशि ज्यादा इन nonspecific प्रतिक्रियाओं से भी बड़ा हो, और हो सकता है प्राइमर लक्ष्य डीएनए अणु के लिए annealing की प्रतियोगिता की सुविधा हो सकती है. यह भी है इसलिए हम पृष्ठभूमि प्रवर्धन देखते हैं, कर सकते हैं और पहले दौर पीसीआर उत्पाद पृष्ठभूमि amplification.It कमी पतला हो गया है महत्वपूर्ण है के लिए पीसीआर प्रतिक्रिया से अधिक प्राइमरों को हटाने क्रम में पीसीआर के दूसरे दौर में पृष्ठभूमि में कमी क्यों है. सभी पीसीआर आधारित प्रयोगों के रूप में, यह महत्वपूर्ण है कि दोहराने अनुक्रम क्षेत्रों, जो मानव और चूहे डीएनए के बहुमत का गठन में स्थित नहीं हैं प्राइमरों डिजाइन. हालांकि नव विकसित हाय - सी पद्धति जीनोम चौड़ा डीएनए संघ के अध्ययन के लिए लागू किया जा सकता है, अधिनियम परख अभी भी ठिकाना विशिष्ट डीएनए बातचीत के अध्ययन के लिए एक प्रभावी तकनीक है.

<!DOCTYPE html PUBLIC "-//W3C//DTD XHTML 1.0 Transitional//EN" "http://www.w3.org/TR/xhtml1/DTD/xhtml1-transitional.dtd">
<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>RPMI1640 medium</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>22400-105</td>
<td>&nbsp;</td>
<tr>
<td>acrylamide</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>15512-023</td>
<td>&nbsp;</td>
<tr>
<td>ATP solution, 10mM</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>AM8110G</td>
<td>&nbsp;</td>
<tr>
<td>fetal bovine serum</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>16000-044</td>
<td>&nbsp;</td>
<tr>
<td>penicillin-streptomycin</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>15140-122</td>
<td>&nbsp;</td>
<tr>
<td>1M Tris pH8.0</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>AM9856</td>
<td>&nbsp;</td>
<tr>
<td>RNase A</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>12091-039</td>
<td>&nbsp;</td>
<tr>
<td>SDS</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>15525-017</td>
<td>&nbsp;</td>
<tr>
<td>urea</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>15505-035</td>
<td>&nbsp;</td>
<tr>
<td>BamH I</td>
<td>&nbsp;</td>
<td>NEB Biolabs</td>
<td>R0136T</td>
<td>&nbsp;</td>
<tr>
<td>Bgl II</td>
<td>&nbsp;</td>
<td>NEB Biolabs</td>
<td>R0144M</td>
<td>&nbsp;</td>
<tr>
<td>Dpn II</td>
<td>&nbsp;</td>
<td>NEB Biolabs</td>
<td>R0543T</td>
<td>&nbsp;</td>
<tr>
<td>Msp I</td>
<td>&nbsp;</td>
<td>NEB Biolabs</td>
<td>R0106S</td>
<td>&nbsp;</td>
<tr>
<td>dNTPs</td>
<td>&nbsp;</td>
<td>NEB Biolabs</td>
<td>N0447L</td>
<td>&nbsp;</td>
<tr>
<td>proteinase K</td>
<td>&nbsp;</td>
<td>NEB Biolabs</td>
<td>P8102S</td>
<td>&nbsp;</td>
<tr>
<td>T4 DNA ligase</td>
<td>&nbsp;</td>
<td>NEB Biolabs</td>
<td>M0202T</td>
<td>&nbsp;</td>
<tr>
<td>37% formaldehyde</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>F8775</td>
<td>&nbsp;</td>
<tr>
<td>Bis-acrylamide</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>146072</td>
<td>&nbsp;</td>
<tr>
<td>dithiothreitol</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>43815</td>
<td>&nbsp;</td>
<tr>
<td>glycine</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>50046</td>
<td>&nbsp;</td>
<tr>
<td>PMSF</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>93482</td>
<td>&nbsp;</td>
<tr>
<td>proteinase inhibitor</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>S8830</td>
<td>&nbsp;</td>
<tr>
<td>Nonidet P-40</td>
<td>&nbsp;</td>
<td>Roche Applied Science</td>
<td>11754599001</td>
<td>&nbsp;</td>
<tr>
<td>KlenTaq1</td>
<td>&nbsp;</td>
<td>Ab peptides</td>
<td>1001</td>
<td>&nbsp;</td>
<tr>
<td>dCTP alpha P32</td>
<td>&nbsp;</td>
<td>PerkinElmer</td>
<td>BLU513H250UC</td>
<td>&nbsp;</td>
<tr>
<td>PTC-100 Thermal Cycler</td>
<td>&nbsp;</td>
<td>MJ Research</td>
<td>mjptc100</td>
<td>&nbsp;</td>
<tr>
<td>Power Supply</td>
<td>&nbsp;</td>
<td>Bio-Rad</td>
<td>164-5056</td>
<td>&nbsp;</td>
<tr>
<td>OmniPAGE Maxi</td>
<td>&nbsp;</td>
<td>Aurogene Life Science</td>
<td>VS20D</td>
<td>&nbsp;</td>
<tr>
<td>Typhoon 9400</td>
<td>&nbsp;</td>
<td>GE Healthcare </td>
<td>63-0055-78</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

associated chromosome trap for identifying long-range dna interactions

आनुवंशिक डीएनए द्वारा इनकोडिंग जानकारी एक जटिल और उच्च विनियमित chromatin संरचना में आयोजित किया जाता है. प्रत्येक गुणसूत्र के एक विशिष्ट क्षेत्र में रह रहे हैं, कि विकास की अवस्था या कोशिका चक्र के अनुसार बदल सकते हैं. जीन अभिव्यक्ति विशेष transcriptional कारखानों में हो सकता है जहां chromatin क्षेत्रों गुणसूत्र के विभिन्न प्रदेशों से बाहर पाश डीएनए खंडों जो अलग या एक ही गुणसूत्र पर गुणसूत्रों दूर पर मौजूद हो सकता है की सह स्थानीयकरण करने के लिए अग्रणी हो सकता है. एसोसिएटेड क्रोमोजोम ट्रैप परख (अधिनियम) एक प्रभावी पद्धति के विस्तार और गुणसूत्र रचना पर कब्जा तकनीक को संशोधित करके इन लंबी दूरी की एक निष्पक्ष फैशन में डीएनए संघों की पहचान प्रदान करता है. अधिनियम परख के लिए यह संभव बनाता है हमें ट्रांस में transcriptional विनियमन के तंत्र की जांच करने के लिए, और परमाणु वास्तुकला का सामान्य और रोग राज्यों के दौरान शरीर क्रिया विज्ञान में जीन अभिव्यक्ति के रिश्ते को समझाने में मदद कर सकते हैं.

Watch this Scientific Journal Video about Associated Chromosome Trap for Identifying Long-range DNA Interactions at JoVE.com

Associated Chromosome Trap for Identifying Long-range DNA Interactions

संबद्ध गुणसूत्र जाल परख (अधिनियम) लंबी दूरी की डीएनए बातचीत की पहचान करने के लिए एक उपन्यास विधि निष्पक्ष है. लंबी दूरी डीएनए बातचीत के लक्षण वर्णन हमें दोनों सामान्य शरीर विज्ञान में और रोगग्रस्त राज्यों में परमाणु वास्तुकला का संबंध जीन की अभिव्यक्ति के लिए निर्धारित करने के लिए अनुमति देगा.

लंबी दूरी की डीएनए सहभागिता की पहचान के लिए एसोसिएटेड क्रोमोजोम जाल

Genetic information encoded by DNA is organized in a complex and highly regulated chromatin structure.  Each chromosome occupies a specific territory, ...

associated-chromosome-trap-for-identifying-long-range-dna-interactions

Research

JoVE Journal

Biology

Split-Ubiquitin Based Membrane Yeast Two-Hybrid (MYTH) System: A Powerful Tool For Identifying Protein-Protein Interactions

The fundamental biological and clinical importance of integral membrane proteins prompted the development of a yeast-based system for the high-throughput identification of protein-protein interactions (PPI) for full-length transmembrane proteins. To this end, our lab developed the split-ubiquitin based Membrane Yeast Two-Hybrid (MYTH) system. This technology allows for the sensitive detection of transient and stable protein interactions using Saccharomyces cerevisiae as a host organism. MYTH takes advantage of the observation that ubiquitin can be separated into two stable moieties: the C-terminal half of yeast ubiquitin (Cub) and the N-terminal half of the ubiquitin moiety (Nub). In MYTH, this principle is adapted for use as a 'sensor' of protein-protein interactions. Briefly, the integral membrane bait protein is fused to Cub which is linked to an artificial transcription factor. Prey proteins, either in individual or library format, are fused to the Nub moiety. Protein interaction between the bait and prey leads to reconstitution of the ubiquitin moieties, forming a full-length 'pseudo-ubiquitin' molecule. This molecule is in turn recognized by cytosolic deubiquitinating enzymes, resulting in cleavage of the transcription factor, and subsequent induction of reporter gene expression. The system is highly adaptable, and is particularly well-suited to high-throughput screening. It has been successfully employed to investigate interactions using integral membrane proteins from both yeast and other organisms.

Split-Ubiquitin Based Membrane Yeast Two-Hybrid MYTH System: A Powerful Tool For Identifying Protein-Protein Interactions

MYTH allows the sensitive detection of transient and stable interactions between proteins that are expressed in the model organism Saccharomyces cerevisiae. It has been successfully applied to study exogenous and yeast integral membrane proteins in order to identify their interacting partners in a high throughput manner.

भाजित Ubiquitin आधार झिल्ली खमीर (मिथक) दो - हाइब्रिड सिस्टम: प्रोटीन, प्रोटीन बातचीत की पहचान करने के लिए एक शक्तिशाली उपकरण

The fundamental biological and clinical importance of integral membrane proteins prompted the development of a yeast-based system for the high-throughput ...

Preparation of Drosophila Polytene Chromosome Squashes for Antibody Labeling

Drosophila has long been a favorite model system for studying the relationship between chromatin structure and gene regulation due to the cytological advantages provided by the giant salivary gland polytene chromosomes of third instar larvae.  In this tissue the chromosomes undergo many rounds of replication in the absence of cell division giving rise to approximately 1000 copies.  The DNA remains aligned after each replicative cycle resulting in greatly enlarged chromosomes that provide a unique opportunity to correlate chromatin morphology with the localization of specific proteins.  Consequently, there has been a high level of interest in defining the epigenetic modifications present at different genes and at different stages of the transcription process. An important tool for such studies is the labeling of polytene chromosomes with antibodies to the enzyme, transcription factor, or histone modification of interest. This video protocol illustrates the squash technique used in the Johansen laboratory to prepare Drosophila polytene chromosomes for antibody labeling.

This video protocol illustrates the squash technique used in the Johansen laboratory to prepare Drosophila polytene chromosomes for antibody labeling.

एंटीबॉडी लेबल के लिए ड्रोसोफिला Polytene क्रोमोजोम Squashes की तैयारी

Drosophila has long been a favorite model system for studying the relationship between chromatin structure and gene regulation due to the cytological ...

Measuring the Bending Stiffness of Bacterial Cells Using an Optical Trap

We developed a protocol to measure the bending rigidity of filamentous rod-shaped bacteria. Forces are applied with an optical trap, a microscopic three-dimensional spring made of light that is formed when a high-intensity laser beam is focused to a very small spot by a microscope's objective lens. To bend a cell, we first bind live bacteria to a chemically-treated coverslip. As these cells grow, the middle of the cells remains bound to the coverslip but the growing ends are free of this restraint. By inducing filamentous growth with the drug cephalexin, we are able to identify cells in which one end of the cell was stuck to the surface while the other end remained unattached and susceptible to bending forces. A bending force is then applied with an optical trap by binding a polylysine-coated bead to the tip of a growing cell. Both the force and the displacement of the bead are recorded and the bending stiffness of the cell is the slope of this relationship.

We present a protocol for bending filamentous bacterial cells attached to a cover-slip surface with an optical trap to measure the cellular bending stiffness.

बैक्टीरियल कोशिकाओं के झुका कठोरता मापने एक ऑप्टिकल जाल का प्रयोग

We developed a protocol to measure the bending rigidity of filamentous rod-shaped bacteria. Forces are applied with an optical trap, a microscopic ...

A Fluorescent Screening Assay for Identifying Modulators of GIRK Channels

G protein-gated inward rectifier K+ (GIRK) channels function as cellular mediators of a wide range of hormones and neurotransmitters and are expressed in the brain, heart, skeletal muscle and endocrine tissue1,2. GIRK channels become activated following the binding of ligands (neurotransmitters, hormones, drugs, etc.) to their plasma membrane-bound, G protein-coupled receptors (GPCRs). This binding causes the stimulation of G proteins (Gi and Go) which subsequently bind to and activate the GIRK channel. Once opened the GIRK channel allows the movement of K+ out of the cell causing the resting membrane potential to become more negative. As a consequence, GIRK channel activation in neurons decreases spontaneous action potential formation and inhibits the release of excitatory neurotransmitters. In the heart, activation of the GIRK channel inhibits pacemaker activity thereby slowing the heart rate.
GIRK channels represent novel targets for the development of new therapeutic agents for the treatment neuropathic pain, drug addiction, cardiac arrhythmias and other disorders3. However, the pharmacology of these channels remains largely unexplored. Although a number of drugs including anti-arrhythmic agents, antipsychotic drugs and antidepressants block the GIRK channel, this inhibition is not selective and occurs at relatively high drug concentrations3.
Here, we describe a real-time screening assay for identifying new modulators of GIRK channels. In this assay, neuronal AtT20 cells, expressing GIRK channels, are loaded with membrane potential-sensitive fluorescent dyes such as bis-(1,3-dibutylbarbituric acid) trimethine oxonol [DiBAC4(3)] or HLB 021-152 (Figure 1). The dye molecules become strongly fluorescent following uptake into the cells (Figure 1). Treatment of the cells with GPCR ligands stimulates the GIRK channels to open. The resulting K+ efflux out of the cell causes the membrane potential to become more negative and the fluorescent signal to decrease (Figure 1). Thus, drugs that modulate K+ efflux through the GIRK channel can be assayed using a fluorescent plate reader. Unlike other ion channel screening assays, such atomic absorption spectrometry4 or radiotracer analysis5, the GIRK channel fluorescent assay provides a fast, real-time and inexpensive screening procedure.

A real-time screening procedure for identifying drugs that interact with G protein-gated inward rectifier K+ (GIRK) channels is described. The assay utilizes membrane potential-sensitive fluorescent dyes to measure GIRK channel activity. This technique is adaptable for use on a number of cell lines.

GIRK चैनल की Modulators की पहचान के लिए एक फ्लोरोसेंट जांच परख

G protein-gated inward rectifier K+ (GIRK) channels function as cellular mediators of a wide range of hormones and neurotransmitters and are expressed in ...

Combining Single-molecule Manipulation and Imaging for the Study of Protein-DNA Interactions

The paper describes the combination of optical tweezers and single molecule fluorescence detection for the study of protein-DNA interaction. The method offers the opportunity of investigating interactions occurring in solution (thus avoiding problems due to closeby surfaces as in other single molecule methods), controlling the DNA extension and tracking interaction dynamics as a function of both mechanical parameters and DNA sequence. The methods for establishing successful optical trapping and nanometer localization of single molecules are illustrated. We illustrate the experimental conditions allowing the study of interaction of lactose repressor (lacI), labeled with Atto532, with a DNA molecule containing specific target sequences (operators) for LacI binding. The method allows the observation of specific interactions at the operators, as well as one-dimensional diffusion of the protein during the process of target search. The method is broadly applicable to the study of protein-DNA interactions but also to molecular motors, where control of the tension applied to the partner track polymer (for example actin or microtubules) is desirable.

Here we describe the instrumentation and methods for detecting single fluorescently-labeled protein molecules interacting with a single DNA molecule suspended between two optically trapped microspheres.

प्रोटीन डीएनए सहभागिता के अध्ययन के लिए एकल अणु हेरफेर और इमेजिंग का मेल

The paper describes the combination of optical tweezers and single molecule fluorescence detection for the study of protein-DNA interaction. The method ...

Real-time Imaging of Single Engineered RNA Transcripts in Living Cells Using Ratiometric Bimolecular Beacons

The growing realization that both the temporal and spatial regulation of gene expression can have important consequences on cell function has led to the development of diverse techniques to visualize individual RNA transcripts in single living cells. One promising technique that has recently been described utilizes an oligonucleotide-based optical probe, ratiometric bimolecular beacon (RBMB), to detect RNA transcripts that were engineered to contain at least four tandem repeats of the RBMB target sequence in the 3&#8217;-untranslated region. RBMBs are specifically designed to emit a bright fluorescent signal upon hybridization to complementary RNA, but otherwise remain quenched. The use of a synthetic probe in this approach allows photostable, red-shifted, and highly emissive organic dyes to be used for imaging. Binding of multiple RBMBs to the engineered RNA transcripts results in discrete fluorescence spots when viewed under a wide-field fluorescent microscope. Consequently, the movement of individual RNA transcripts can be readily visualized in real-time by taking a time series of fluorescent images. Here we describe the preparation and purification of RBMBs, delivery into cells by microporation and live-cell imaging of single RNA transcripts.

Ratiometric bimolecular beacons (RBMBs) can be used to image single engineered RNA transcripts in living cells. Here, we describe the preparation and purification of RBMBs, delivery of RBMBs into cells by microporation and fluorescent imaging of single RNA transcripts in real-time.

Ratiometric bimolecular संकेतकों का उपयोग जीवित कोशिकाओं में एकल इंजीनियर शाही सेना टेप के वास्तविक समय इमेजिंग

The growing realization that both the temporal and spatial regulation of gene expression can have important consequences on cell function has led to the ...

Combined DNA-RNA Fluorescent In situ Hybridization (FISH) to Study X Chromosome Inactivation in Differentiated Female Mouse Embryonic Stem Cells

Fluorescent in situ hybridization (FISH) is a molecular technique which enables the detection of nucleic acids in cells. DNA FISH is often used in cytogenetics and cancer diagnostics, and can detect aberrations of the genome, which often has important clinical implications. RNA FISH can be used to detect RNA molecules in cells and has provided important insights in regulation of gene expression. Combining DNA and RNA FISH within the same cell is technically challenging, as conditions suitable for DNA FISH might be too harsh for fragile, single stranded RNA molecules. We here present an easily applicable protocol which enables the combined, simultaneous detection of Xist RNA and DNA encoded by the X chromosomes. This combined DNA-RNA FISH protocol can likely be applied to other systems where both RNA and DNA need to be detected.

Combined DNA-RNA Fluorescent In situ Hybridization FISH to Study X Chromosome Inactivation in Differentiated Female Mouse Embryonic Stem Cells

Fluorescent in situ hybridization (FISH) allows the detection of nucleic acids in their native environment within cells. We here describe a protocol for the combined, simultaneous detection of RNA and DNA by means of FISH, which can be used to study X chromosome inactivation in mouse embryonic stem cells.

संयुक्त डीएनए शाही सेना फ्लोरिसेंट<em&gt; सीटू</em&gt; संकरण (मछली) विभेदित महिला माउस भ्रूणीय स्टेम कोशिकाओं में एक्स गुणसूत्र निष्क्रियता अध्ययन करने के लिए

Fluorescent in situ hybridization (FISH) is a molecular technique which enables the detection of nucleic acids in cells. DNA FISH is often used in ...

Chromatin Immunoprecipitation Assay for the Identification of Arabidopsis Protein-DNA Interactions In Vivo

Intricate gene regulatory networks orchestrate biological processes and developmental transitions in plants. Selective transcriptional activation and silencing of genes mediate the response of plants to environmental signals and developmental cues. Therefore, insights into the mechanisms that control plant gene expression are essential to gain a deep understanding of how biological processes are regulated in plants. The chromatin immunoprecipitation (ChIP) technique described here is a procedure to identify the DNA-binding sites of proteins in genes or genomic regions of the model species Arabidopsis thaliana. The interactions with DNA of proteins of interest such as transcription factors, chromatin proteins or posttranslationally modified versions of histones can be efficiently analyzed with the ChIP protocol. This method is based on the fixation of protein-DNA interactions in vivo, random fragmentation of chromatin, immunoprecipitation of protein-DNA complexes with specific antibodies, and quantification of the DNA associated with the protein of interest by PCR techniques. The use of this methodology in Arabidopsis has contributed significantly to unveil transcriptional regulatory mechanisms that control a variety of plant biological processes. This approach allowed the identification of the binding sites of the Arabidopsis chromatin protein EBS to regulatory regions of the master gene of flowering FT. The impact of this protein in the accumulation of particular histone marks in the genomic region of FT was also revealed through ChIP analysis.

Chromatin Immunoprecipitation Assay for the Identification of Arabidopsis Protein-DNA Interactions In Vivo

Chromatin immunoprecipitation is a powerful technique for the identification of DNA binding sites of Arabidopsis proteins in vivo. This procedure includes chromatin cross-linking and fragmentation, immunoprecipitation with selective antibodies against the protein of interest, and qPCR analysis of bound DNA. We describe a simple ChIP assay optimized for Arabidopsis plants.

एराबिडोप्सिस प्रोटीन डीएनए बातचीत की पहचान के लिए chromatin immunoprecipitation परख<em&gt; Vivo</em

Intricate gene regulatory networks orchestrate biological processes and developmental transitions in plants. Selective transcriptional activation and ...

DamID-seq: Genome-wide Mapping of Protein-DNA Interactions by High Throughput Sequencing of Adenine-methylated DNA Fragments

The DNA adenine methyltransferase identification (DamID) assay is a powerful method to detect protein-DNA interactions both locally and genome-wide. It is an alternative approach to chromatin immunoprecipitation (ChIP). An expressed fusion protein consisting of the protein of interest and the E. coli DNA adenine methyltransferase can methylate the adenine base in GATC motifs near the sites of protein-DNA interactions. Adenine-methylated DNA fragments can then be specifically amplified and detected. The original DamID assay detects the genomic locations of methylated DNA fragments by hybridization to DNA microarrays, which is limited by the availability of microarrays and the density of predetermined probes. In this paper, we report the detailed protocol of integrating high throughput DNA sequencing into DamID (DamID-seq). The large number of short reads generated from DamID-seq enables detecting and localizing protein-DNA interactions genome-wide with high precision and sensitivity. We have used the DamID-seq assay to study genome-nuclear lamina (NL) interactions in mammalian cells, and have noticed that DamID-seq provides a high resolution and a wide dynamic range in detecting genome-NL interactions. The DamID-seq approach enables probing NL associations within gene structures and allows comparing genome-NL interaction maps with other functional genomic data, such as ChIP-seq and RNA-seq.

We describe herein an assay by coupling DNA adenine methyltransferase identification (DamID) to high throughput sequencing (DamID-seq). This improved method provides a higher resolution and a wider dynamic range, and allows analyzing DamID-seq data in conjunction with other high throughput sequencing data such as ChIP-seq, RNA-seq, etc.

DamID-सेक: Adenine-methylated डीएनए टुकड़े के उच्च throughput अनुक्रमण द्वारा प्रोटीन डीएनए सहभागिता के जीनोम चौड़ा मानचित्रण

The DNA adenine methyltransferase identification (DamID) assay is a powerful method to detect protein-DNA interactions both locally and genome-wide. It is ...

Amplification, Next-generation Sequencing, and Genomic DNA Mapping of Retroviral Integration Sites

Retroviruses exhibit signature integration preferences on both the local and global scales. Here, we present a detailed protocol for (1) generation of diverse libraries of retroviral integration sites using ligation-mediated PCR (LM-PCR) amplification and next-generation sequencing (NGS), (2) mapping the genomic location of each virus-host junction using BEDTools, and (3) analyzing the data for statistical relevance. Genomic DNA extracted from infected cells is fragmented by digestion with restriction enzymes or by sonication. After suitable DNA end-repair, double-stranded linkers are ligated onto the DNA ends, and semi-nested PCR is conducted using primers complementary to both the long terminal repeat (LTR) end of the virus and the ligated linker DNA. The PCR primers carry sequences required for DNA clustering during NGS, negating the requirement for separate adapter ligation. Quality control (QC) is conducted to assess DNA fragment size distribution and adapter DNA incorporation prior to NGS. Sequence output files are filtered for LTR-containing reads, and the sequences defining the LTR and the linker are cropped away. Trimmed host cell sequences are mapped to a reference genome using BLAT and are filtered for minimally 97% identity to a unique point in the reference genome. Unique integration sites are scrutinized for adjacent nucleotide (nt) sequence and distribution relative to various genomic features. Using this protocol, integration site libraries of high complexity can be constructed from genomic DNA in three days. The entire protocol that encompasses exogenous viral infection of susceptible tissue culture cells to integration site analysis can therefore be conducted in approximately one to two weeks. Recent applications of this technology pertain to longitudinal analysis of integration sites from HIV-infected patients.

We describe a protocol for amplifying retroviral integration sites from the genomic DNA of infected cells, sequencing the amplified virus-host junctions, and then mapping these sequences to a reference genome. We also describe techniques to quantify the distribution of integration sites relative to various genomic annotations using BEDTools.

रेट्रोवायरल एकता स्थलों की प्रवर्धन, अगली पीढ़ी के अनुक्रमण, और जीनोमिक डीएनए का मिलान

Retroviruses exhibit signature integration preferences on both the local and global scales. Here, we present a detailed protocol for (1) generation of ...