Name: analysis of line-1 retrotransposition at the single nucleus level
Uploaded: 2016-04-23T14:00:00
Description: Watch this Scientific Journal Video about Analysis of LINE-1 Retrotransposition at the Single Nucleus Level at JoVE.com

This work was supported in part by grants from the National Institute of Environmental Health Sciences (ES014443 and ES017274) and AstraZeneca to KSR.

HINWEIS: Alle Schritte sollten bei Raumtemperatur durchgeführt werden, wenn nicht anders angegeben. Bitte beachten Sie Reagenzien für Details auf, wie einzelne Reagenzien herzustellen. 1. Kennzeichnung L1 Probes HINWEIS: Die Sonden können durch chemische oder PCR-Kennzeichnung versehen werden. <ol><li> chemische Markierung <ol><li> Machen 0,7-1% Agarosegel in Tris-Acetat-EDTA (TAE) -Puffer, Wärme in einer Mikrowelle, bis sie geschm...

<ol>
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V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+frequency+retrotransposition+in+cultured+mammalian+cells.">High frequency retrotransposition in cultured mammalian cells.</a> Cell. 87, 917-927 (1996).</li><li>Bojang, P., Anderton, M. J., Roberts, R. A., Ramos, K. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=De+novo+LINE-1+retrotransposition+in+HepG2+cells+preferentially+targets+gene+poor+regions+of+chromosome+13.">De novo LINE-1 retrotransposition in HepG2 cells preferentially targets gene poor regions of chromosome 13.</a> Genomics. 104, 96-104 (2014).</li><li>Rozen, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Abundant+gene+conversion+between+arms+of+palindromes+in+human+and+ape+Y+chromosomes.">Abundant gene conversion between arms of palindromes in human and ape Y chromosomes.</a> Nature. 423, 873-876 (2003).</li><li>Graves, J. A., Koina, E., Sankovic, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+the+gene+content+of+human+sex+chromosomes+evolved.">How the gene content of human sex chromosomes evolved.</a> Curr Op Gen Develop. 16, 219-224 (2006).</li><li>Leem, Y. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retrotransposon+Tf1+is+targeted+to+Pol+II+promoters+by+transcription+activators.">Retrotransposon Tf1 is targeted to Pol II promoters by transcription activators.</a> Mol Cell. 30, 98-107 (2008).</li><li>Guo, Y., Levin, H. 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Methodiken wie Sequenzierung ganzer Genome, inverse PCR und Southern - Blotting wurden zu studieren L1 Retrotransposition eingesetzt. Obwohl diese Methoden äußerst wertvoll sind , bei der Suche nach dem L1 Einfügungen treten innerhalb von Genomen, eine verwirrende Herausforderung für alle von ihnen ist die Notwendigkeit für die In-silico - Programmierung zu Sequenzen wieder zusammen. Die FISH - Methodik hier beschrieben entwickelt , um diese Methoden zu ergänzen, insbesondere im Fall von...

Menschliche Lange streute Kern Element-1 (Linie-1 oder L1) ist eine autonome mobile Element , das durch eine im Genom ausbreitet &quot;Kopieren und Einfügen&quot; Retrotransposition Mechanismus. Ein typisches menschliches L1 ~ 6 kb lang und besteht aus einer 5&#39; - UTR (untranslatierte Region), die als Promotor dient, zwei offene Leserahmen (ORFs): L1 -ORF1 und L1 -ORF2 und eine 3&#39;UTR mit einem polyA . Schwanz L1 -ORF1 Protein hat drei verschiedene Bereiche: eine Spule-Coil - Domäne, RNA - Erkennungsmotiv und C-terminale Domäne, während L1 -ORF2 Protein Endonuklease hat, reverse Transkriptase und Cystein reiche Domänen 1.2.3.4.5. L1 -ORF1 zeigt Nukleinsäure Chaperon - Aktivitäten, während L1 -ORF2 enzymatischen Aktivitäten bietet, wobei beide für Retrotransposition benötigten Proteine ​​1,2,6. Ein Zyklus von L1 Retrotransposition beginnt mit der Transkription von der 5&#39;UTR von L1 </em&gt; bicistronischen mRNA durch RNA-Polymerase II, Translokation in das Cytoplasma und Translation. Im Zytoplasma, L1 -ORF1 weist entweder cis -Bindung , wo es seine eigene RNA oder trans -Bindung Pakete , wo es andere RNAs Pakete (dh Sinus- / SVAS / pseudogenes) 7 ein Ribonukleoprotein Partikel (RNP) mit L1 -ORF2p zu bilden, 8. RNPs Translokation in den Zellkern , wo die Endonuklease - Aktivität von L1 -ORF2p Scharten genomische DNA (gDNA) eine OH zu belichten - Gruppe 9, die wiederum durch die reverse Transkriptase zu primen verwendet wird und synthesize RNA zu DNA vom 3&#39; - Ende umzukehren. Während der Rückwärts Synthese wird der zweite DNA - Strang 7-20 bp von der ursprünglichen nick Website geklaut zu versetzten Pausen schaffen , die die Signatur L1 Insertionssequenzen (zB TTTTAA) genannt Zielstelle Vervielfältigungen (TSD) 9,10 zu bilden , gefüllt sind. Dieser Vorgang wird als Ziel prime reverse Transkription (TPRT) bekannt und führt zu insertiauf der vollständigen oder abgeschnittene Kopien von L1 / andere DNAs mit TSD an beiden Enden der eingefügten Sequenz. L1 Retrotransposition auch durch TPRT artigen nicht-homologe end-joining in einigen Zelltypen 11 vermittelt zu werden hat sich gezeigt. Der L1 - Vektor Retrotransposition in unseren Studien verwendeten nicht-episomale und besteht aus getaggt L1 -ORF1 &amp; 2p angetrieben durch kombinierte CMV-Promotoren L1-5&#39;UTR (Abbildung 1) 12. Frühere Versionen dieses Konstrukts wurden 13,14,15 in Studien unter Verwendung von Hefe und menschlichen Zellkulturen beschrieben. Zwei verschiedene CMV-Promotoren in der 5 &#39;angeordnet und 3&#39;-Enden des Vektors, mit dem 3&#39;-Ende in umgekehrter Ausrichtung angeordnet Expression von Neomycin nach dem Spleißen und Integration zu fahren. Der Retrotransposition Indikator Kassette an dem 3&#39; - Ende besteht aus einem Neomycin - Gen insertiert Antisense zu L1 -ORFs und durch eine glob inaktiver durch Trennung in zwei Hälften gemachtin Intron mit gespleißten Donor (SD) und gespleißt Akzeptor (SA) -Stellen (Abbildung 1). Bei der Integration in ein Chromosom, wird L1 von dem gemeinsamen Promotor transkribiert , um eine mRNA produzieren , die von einer bicistronischen mRNA und inaktive Neomycin mRNA besteht. Während RNA Verarbeitung wird das Globin-Intron aus dem Neomycin-Gen gespleißt eine voll funktionsfähige Neomycin-Gen wiederherzustellen. Die Hybrid - mRNA wird in ein RNP in cis verpackt und in den Zellkern transloziert , wo es in das Genom entweder in voller Länge oder abgeschnittene Einfügungen Verwendung TPRT integriert ist. Hier beschreiben wir eine Methode , die Fluoreszenz - in - situ - Hybridisierung (FISH) mit Sonden verwendet speziell auf die gespleißten Neomycin - Gen gerichtet (Sneo) an der einzigen Kern Ebene L1 -retrotransposiition Muster und Einbringungsraten zu verfolgen. Die Effizienz und Spezifität des Nachweises wurde unter Verwendung von Retrotransposition kompetenten und inkompetenten Konstrukte und Sonden bestätigt detect Sneo oder das Neomycin und Intron - Übergang Globin - 16. Diese Methodologie macht einige der Mängel der Zellkultur-Assays Retrotransposition, beispielsweise mehrere Einfügungen, kolonieBeständigkeit und günstige clone Expansion.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Labeling Probes</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Go Tag DNA polymerase</td>
			<td>Promega</td>
			<td>M3178</td>
			<td></td>
		</tr>
		<tr>
			<td>GO Tag 10X colorless buffer</td>
			<td>Promega</td>
			<td>M3178</td>
			<td></td>
		</tr>
		<tr>
			<td>Individual dNTP</td>
			<td>Sigma</td>
			<td>DNTP10</td>
			<td>Adjust the concentration of dATP, dGTP, dCTP to 2mM and dTTP to 1.5mM&#160; in nuclease free water.</td>
		</tr>
		<tr>
			<td>&#160;dUTP-16-Biotin (0.5mM)</td>
			<td>Roche</td>
			<td>11093070910</td>
			<td></td>
		</tr>
		<tr>
			<td>dUTP-FITC (0.5mM)</td>
			<td>Thermo Scientific</td>
			<td>R0101</td>
			<td></td>
		</tr>
		<tr>
			<td>dUTP-CY3 (0.5mM)</td>
			<td>Sigma</td>
			<td>GEPA53022</td>
			<td></td>
		</tr>
		<tr>
			<td>MIRUS FISH Labeling Kit</td>
			<td>MIRUS</td>
			<td>MIR3625</td>
			<td></td>
		</tr>
		<tr>
			<td>MIRUS FISH Labeling Kit</td>
			<td>MIRUS</td>
			<td>MIR3225</td>
			<td></td>
		</tr>
		<tr>
			<td>NucleoSpin PCR Clean-Up kit&#160;</td>
			<td>Qiagen</td>
			<td>1410/0030</td>
			<td>Any PCR clean reagent can be used in place of NucleoSpin PCR Clean Up Kit</td>
		</tr>
		<tr>
			<td>PCR Machine</td>
			<td></td>
			<td></td>
			<td>Any Thermocycler machine can be used to amplify the label product.</td>
		</tr>
		<tr>
			<td>Agarose</td>
			<td>Sigma</td>
			<td>A9539</td>
			<td></td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Preparing chromosome Spreads.</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>&#160;&#160;&#160;&#160; Colcemid&#160;</td>
			<td>Life Technologies&#160;</td>
			<td>15212-012</td>
			<td>Add Colcemid directly to growth media&#160; to a final concentration of 0.4 &#181;g/mL</td>
		</tr>
		<tr>
			<td>1M KCl</td>
			<td>Sigma</td>
			<td>P9541</td>
			<td>Dilute 1M KCl solution to 75 mM solution to make Hypotonic Solution</td>
		</tr>
		<tr>
			<td>Carnoy Fixative Solution</td>
			<td></td>
			<td></td>
			<td>Mix 3 volumes of methanol and 1 volume of acetic acid to make Carnoy Fixative Solution. Make Fresh everytime.</td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td>Sigma</td>
			<td>322415</td>
			<td></td>
		</tr>
		<tr>
			<td>Acetic acid</td>
			<td>Sigma</td>
			<td>320099</td>
			<td></td>
		</tr>
		<tr>
			<td>Hoechst-33342</td>
			<td>Thermo Scientific</td>
			<td>62249</td>
			<td>Dissolve Hoechst-33342 in PBS to final concentration of 1 mg/mL.</td>
		</tr>
		<tr>
			<td>Diamond Point Marker</td>
			<td>Thermo Scientific</td>
			<td>750</td>
			<td></td>
		</tr>
		<tr>
			<td>Frosted Slides</td>
			<td>Thermo Scientific</td>
			<td>2951-001</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin-EDTA (0.25%)</td>
			<td>Life Technologies&#160;</td>
			<td>R001100</td>
			<td>To detech cells, incubate cell in Trypsin for 5 mins and inactive with equal volume of complete media</td>
		</tr>
		<tr>
			<td>Cover slides&#160;</td>
			<td>VWR</td>
			<td>48366067</td>
			<td></td>
		</tr>
		<tr>
			<td>Coplin Jars</td>
			<td>Thermo Scientific</td>
			<td>107</td>
			<td></td>
		</tr>
		<tr>
			<td>&#160;Heat Block</td>
			<td></td>
			<td></td>
			<td>Any heat block can be used for this purpose, though blocks fitted for heating slides are recommended.</td>
		</tr>
		<tr>
			<td>&#160;Beaker (600ml)</td>
			<td>Sigma</td>
			<td>CLS1003600</td>
			<td>Fill the beaker with water, heat to boil, use the hot steam to burst chromosomes.</td>
		</tr>
		<tr>
			<td>Nikon Microscope</td>
			<td>Nikon</td>
			<td>125690</td>
			<td>Any microscope can be used to look at spread quality.</td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Reagents and Materials for FISH</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Trisodium citrate</td>
			<td>Sigma</td>
			<td>S1804</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Chroride</td>
			<td>Sigma</td>
			<td>S3014</td>
			<td></td>
		</tr>
		<tr>
			<td>Formamide</td>
			<td>Sigma</td>
			<td>F9037</td>
			<td></td>
		</tr>
		<tr>
			<td>Tween-20</td>
			<td>Sigma</td>
			<td>F1379</td>
			<td></td>
		</tr>
		<tr>
			<td>Detran Sulfate</td>
			<td>Sigma</td>
			<td>D8906</td>
			<td></td>
		</tr>
		<tr>
			<td>SDS</td>
			<td>Sigma</td>
			<td>L3771</td>
			<td></td>
		</tr>
		<tr>
			<td>Salmon Sperm DNA</td>
			<td>Life Technologies&#160;</td>
			<td>15632-011</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin (BSA)</td>
			<td>Sigma</td>
			<td>A8531</td>
			<td></td>
		</tr>
		<tr>
			<td>HCl (N)</td>
			<td>Sigma</td>
			<td>38283</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethyl Alcohol</td>
			<td>Sigma</td>
			<td>459844</td>
			<td></td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td>Sigma</td>
			<td>322415</td>
			<td></td>
		</tr>
		<tr>
			<td>DPBS</td>
			<td>Life Technologies&#160;</td>
			<td>14190-250</td>
			<td></td>
		</tr>
		<tr>
			<td>NaOH</td>
			<td>Sigma</td>
			<td>S8045</td>
			<td></td>
		</tr>
		<tr>
			<td>Rnase A</td>
			<td>Sigma</td>
			<td>R4642</td>
			<td></td>
		</tr>
		<tr>
			<td>Pepsin</td>
			<td>Sigma</td>
			<td>P6887</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde (PFA)</td>
			<td>Sigma</td>
			<td>P6148</td>
			<td></td>
		</tr>
		<tr>
			<td>Hoechst-33342</td>
			<td>Thermo Scientific</td>
			<td>62249</td>
			<td>Dissolve Hoechst-33342 in PBS to final concentration of 1 mg/mL.</td>
		</tr>
		<tr>
			<td>Rubber Cement/Cytobond Sealant&#160;</td>
			<td></td>
			<td>2020-00-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Seven Coplin Jars</td>
			<td>Thermo Scientific</td>
			<td>107</td>
			<td></td>
		</tr>
		<tr>
			<td>&#160; Dark Humidified chamber</td>
			<td>Sigma</td>
			<td>CLS2551</td>
			<td></td>
		</tr>
		<tr>
			<td>Cover slides&#160;</td>
			<td>VWR</td>
			<td>48366067</td>
			<td></td>
		</tr>
		<tr>
			<td>Dry Digital Heat Block</td>
			<td>VWR</td>
			<td>13259</td>
			<td></td>
		</tr>
		<tr>
			<td>&#160;Fluorescence Microscope</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>20X Saline-Sodium Citrate (20X SSC)</td>
			<td></td>
			<td></td>
			<td>Combine 175 g of NaCl, and 88.2 g of trisodium citrate with 800 mL of molecular grade H2O. Stir while adjusting to pH 7. Once the all salts dissolve, adjust the volume to 1 L and filter through 0.22 &#181;m Filter paper. Store at 4 &#176;C.</td>
		</tr>
		<tr>
			<td>&#160;1mg/mL of Rnase A</td>
			<td></td>
			<td></td>
			<td>Dilute 20X SSC buffer to 2X SSC buffer. Dissolve RNase A in 2X SSC buffer to a final concentration of 1 mg/mL. Make fresh solution every time.</td>
		</tr>
		<tr>
			<td>1% Pepsin</td>
			<td></td>
			<td></td>
			<td>Dissolve&#160; pepsin (W/V) in 10 mM HCl to a final concentration of 1 % solution. Make fresh every time.</td>
		</tr>
		<tr>
			<td>4% Paraformaldehyde (4% PFA)</td>
			<td></td>
			<td></td>
			<td>Weigh 4.0 g of PFA in fume hood, add 50 mL of 1xPBS, heat to 60 &#176;C while stirring and adjust the pH with drops of NaOH until all PFA dissolves and the solution becomes clear. Adjust the volume to 100 mL, filter through 0.22 &#181;m Filter paper and store 5 mL aliquots at -20 &#176;C. Thaw aliquot of PFA at 37 &#176;C for 10-15 min for subsequent uses.&#160; Adjust filter size depending on amount of paraformaldehyde.</td>
		</tr>
		<tr>
			<td>Wash Buffer</td>
			<td></td>
			<td></td>
			<td>Combine 100 mL of Formamide (20%) with 2.5 mL of 20X SSC, adjust to pH 7 with 1.0 M HCl and bring the volume to 500 mL with molecular grade H2O.</td>
		</tr>
		<tr>
			<td>Hybridization Buffer</td>
			<td></td>
			<td></td>
			<td>Combine 50% formamide, 10% dextran sulfate, 0.1% SDS, and 300 ng/mL Salmon Sperm DNA in 2X SSC buffer.&#160; Amount of Formamide determine how focused chromatids are, titrate the amount&#160;</td>
		</tr>
		<tr>
			<td>Detection Buffer</td>
			<td></td>
			<td></td>
			<td>Dilute 20X SSC buffer to 4X and add 0.2% Tween-20 to make detection buffer.</td>
		</tr>
		<tr>
			<td>Blocking Buffer</td>
			<td></td>
			<td></td>
			<td>Combine 5% bovine serum albumin (BSA) with 0.2% Tween-20 in 4X SSC buffer.</td>
		</tr>
		<tr>
			<td>Dehydrating Solution</td>
			<td></td>
			<td></td>
			<td>Make 70%, 80%, and 95% ethanol solutions.</td>
		</tr>
	</tbody>
</table>

analysis of line-1 retrotransposition at the single nucleus level

Long interspersed nuclear element-1 (Line-1 or L1) accounts for approximately 17% of the DNA present in the human genome. While the majority of L1s are inactive due to 5' truncations, ~80-100 of these elements remain retrotransposition competent and propagate to different locations throughout the genome via RNA intermediates. While older L1s are believed to target AT rich regions of the genome, the chromosomal targets of newer, more active L1s remain poorly defined. Here we describe fluorescence in situ hybridization (FISH) methodology that can be used to track patterns of L1 insertion and rates of ectopic L1 incorporation at the single nucleus level. In these experiments, fluorescein isothiocyanate/cyanine-3 (FITC/CY3) labeled neomycin probes were employed to track L1 retrotransposition in vitro in HepG2 cells stably expressing ectopic L1. This methodology prevents errors in the estimation of rates of retrotransposition posed by toxicity and account for the occurrence of multiple insertions into a single nucleus.

Ein schematisches Diagramm des L1 Retrotransposition Vektors ist in Abbildung 1 dargestellt. Der Vektor besteht aus einem Neomycin - Gen in Antisense - Orientierung zu L1 ORFs , die durch ein Globin - Intron in Sense - Orientierung und sandwichartig von SD und SA Seiten unterbrochen ist. Wenn stabil in ein Chromosom integriert ist L1 mRNA aus dem kombinierten CMV transkribiert und L1-5&#39;UTR Promotor (Abbildung 1). Während R...

Watch this Scientific Journal Video about Analysis of LINE-1 Retrotransposition at the Single Nucleus Level at JoVE.com

Analysis of LINE-1 Retrotransposition at the Single Nucleus Level

Analyse von LINE-1-Retrotransposition im Einzel Nucleus Ebene

Hier beschäftigen wir FISH - Methodik LINE-1 - Retrotransposition an der einzelnen Kerne Ebene in Chromosom Spreads von HepG2 - Zelllinien stabil synthetischen LINE-1 exprimieren , zu verfolgen.

Long interspersed nuclear element-1 (Line-1 or L1) accounts for approximately 17% of the DNA present in the human genome. While the majority of L1s are ...

Research

JoVE Journal

Biology

Method for Measurement of Viral Fusion Kinetics at the Single Particle Level

Methode zur Messung des Viral Fusion Kinetics auf der Single Particle Stufe

Membrane fusion is an essential step during entry of enveloped viruses into cells. Conventional fusion assays typically report on a large number of fusion ...

Forschung

Biologie

Direct Analysis of Single Cells by Mass Spectrometry at Atmospheric Pressure

Direkte Analyse von Einzelzellen durch Massenspektrometrie bei Atmosphärendruck

Analysis of biochemicals in single cells is important for understanding cell metabolism, cell cycle, adaptation, disease states, etc. Even the same cell ...

Studying Proteolysis of Cyclin B at the Single Cell Level in Whole Cell Populations

Studieren Proteolyse von Cyclin B an der Single Cell Level in Whole Cell Populationen

Equal distribution of chromosomes between the two daughter cells during cell division is a prerequisite for guaranteeing genetic stability 1. Inaccuracies ...

Efficient Agroinfiltration of Plants for High-level Transient Expression of Recombinant Proteins

Effiziente Agroinfiltration von Pflanzen für High-Level-Transient Expression von rekombinanten Proteinen

Mammalian cell culture is the major platform for commercial production of human vaccines and therapeutic proteins. However, it cannot meet the increasing ...

Analysis of Global RNA Synthesis at the Single Cell Level following Hypoxia

Analyse der globalen RNA-Synthese auf der Einzelzellebene folgende Hypoxie

Hypoxia or lowering of the oxygen availability is involved in many physiological and pathological processes. At the molecular level, cells initiate a ...

CUBIC Protocol Visualizes Protein Expression at Single Cell Resolution in Whole Mount Skin Preparations

CUBIC Protokoll Visualisiert Expression Protein bei Single Cell Resolution in Ganze Berge Vorbereitung der Haut

The skin is essential for our survival. The outer epidermal layer consists of the interfollicular epidermis, which is a stratified squamous epithelium ...

Analysis of Beta-cell Function Using Single-cell Resolution Calcium Imaging in Zebrafish Islets

Analyse der Beta-Zellfunktion mit einzelligen Auflösung Kalzium Imaging im Zebrafisch Inselchen

Pancreatic beta-cells respond to increasing blood glucose concentrations by secreting the hormone insulin. The dysfunction of beta-cells leads to ...

Visualizing the Interaction Between the Qdot-labeled Protein and Site-specifically Modified &#955; DNA at the Single Molecule Level

Visualizing the Interaction Between the Qdot-labeled Protein and Site-specifically Modified ÃƒÆ’Ã…Â½Ãƒâ€šÃ‚Â» DNA at the Single Molecule Level

Visualisierung der Interaktion zwischen dem Qdot-markierten Protein und ortspezifisch modifizierte λ-DNA auf der Einzelmolekül-Ebene

The fluorescence microscopy&#160;has made great contributions in dissecting the mechanisms of complex biological processes at the single molecule level. ...

Probing mRNA Kinetics in Space and Time in Escherichia coli using Two-Color Single-Molecule Fluorescence In Situ Hybridization

Probing mRNA Kinetics in Space and Time in Escherichia coli using Two-Color Single-Molecule Fluorescence In Situ Hybridization

Probing mRNA Kinetics in Space and Time in Escherichia coli mit Zwei-Farben-Einmolekülfluoreszenz in Situ-Hybridisierung

Single-molecule fluorescence in situ hybridization (smFISH) allows for counting the absolute number of mRNAs in individual cells. Here, we describe an ...