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.

NOTA: Todas las etapas debe llevarse a cabo a temperatura ambiente a menos que se especifique lo contrario. Por favor refiérase a la sección Reactivos para obtener más información sobre cómo preparar reactivos individuales. 1. Etiquetado L1 Sondas NOTA: Las sondas pueden marcarse mediante marcaje químico o PCR. <ol><li> etiquetado químico <ol><li> Hacer 0,7-1% en gel de agarosa en tampón Tris-acetato-EDTA (TAE), el calor en el ...

<ol>
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Metodologías como la secuenciación del genoma completo, PCR inversa y transferencia de Southern se han empleado para estudiar L1 retrotransposition. A pesar de estas metodologías son extremadamente valiosos en la localización donde se producen L1 inserciones dentro de los genomas, un desafío de confusión para todos ellos es la necesidad de la programación en silico para volver a montar secuencias. La metodología FISH aquí descrito está diseñado para complementar estos métodos, espe...

Humana largos períodos de actividad nuclear Elemento-1 (Line-1 o L1) es un elemento móvil autónomo que se propaga dentro del genoma a través de un &quot;copiar y pegar&quot; mecanismo de retrotransposition. A L1 humana típica es ~ 6 kb de largo y consiste en una 5&#39;UTR (región no traducida) que sirve como un promotor, dos marcos de lectura abierta (ORFs): L1 y L1 -ORF1 -ORF2, y una 3&#39;UTR con un polyA . cola de la proteína L1 -ORF1 tiene tres dominios distintos: un dominio de bobina de la bobina, el reconocimiento de ARN motivo y dominio C-terminal, mientras que la proteína L1 -ORF2 tiene endonucleasa, la transcriptasa inversa y la cisteína dominios ricos en 1.2.3.4.5. L1 -ORF1 exhibe actividad chaperona de ácidos nucleicos, mientras que L1 -ORF2 ofrece actividades enzimáticas, con las dos proteínas necesarias para retrotransposition 1,2,6. Un ciclo de L1 retrotransposition comienza con la transcripción de la 5&#39;UTR de L1 </em&gt; mRNA bicistrónico por la ARN polimerasa II, la translocación en el citoplasma, y ​​la traducción. En el citoplasma, L1 -ORF1 exposiciones cualquiera de unión a cis donde los paquetes de su propio ARN o unión a trans donde los paquetes de otros ARN (es decir, de seno / EASV / pseudogenes) para formar una partícula de ribonucleoproteína (RNP) con L1 -ORF2p 7, 8. RNPs translocan en el núcleo donde la actividad de endonucleasa de nicks L1 -ORF2p DNA genómico (gDNA) para exponer un OH - grupo 9, que es a su vez usada por la transcriptasa inversa para cebar y revertir RNA sintetizar al ADN a partir del extremo 3 &#39;. Durante la síntesis inversa, la segunda cadena de ADN se mella 7-20 pb desde el sitio original nick para crear pausas escalonadas que están llenos para formar las secuencias de inserción L1 de firma (por ejemplo, TTTTAA) llamados objetivo sitio duplicaciones (TSD) 9,10. Este proceso se conoce como la transcripción inversa objetivo prioritario (TPRT) y conduce a insertien de copias completas o truncadas de L1 / otros ADN con DTE en ambos extremos de la secuencia insertada. L1 retrotransposition También se ha demostrado que es mediado a través de TPRT-como unión de extremos en algunos tipos de células 11 no homóloga. El vector de L1 retrotransposition empleado en nuestros estudios es no episomales y consta de etiquetado L1 -ORF1 y 2p impulsada por los promotores de CMV-L1-5&#39;UTR combinados (Figura 1) 12. Las versiones anteriores de esta construcción se han descrito en los estudios que utilizan levadura y cultivos de células humanas 13,14,15. Dos promotores CMV distintos situados en el 5 &#39;y 3&#39; del vector, con el extremo 3 &#39;colocado en orientación inversa para conducir la expresión de la neomicina después de empalme y la integración. El casete indicador de retrotransposición en el extremo 3 &#39;se compone de un gen insertado antisentido neomicina a L1 -ORFs y vuelve inactivo por separación en dos mitades por una globen el intrón con donante empalmado (SD) y sitios aceptores de corte y empalme (SA) (Figura 1). Tras la integración en un cromosoma, L1 es transcrito a partir del promotor común para producir un mRNA que consiste en un mRNA bicistrónico y mRNA neomicina inactivo. Durante el procesamiento del ARN, el intrón de globina se empalma fuera del gen de la neomicina para restaurar un gen de la neomicina totalmente funcional. El ARNm híbrido se empaqueta en un RNP en cis y transloca en el núcleo cuando se integre en el genoma, ya sea como de longitud completa o truncadas inserciones utilizando TPRT. Aquí se describe una metodología que utiliza hibridación in situ fluorescente (FISH) con sondas dirigidas específicamente al gen de la neomicina empalmado (SNEO) para rastrear los patrones -retrotransposiition L1 y las tasas de inserción a nivel de núcleo único. La eficiencia y la especificidad de la detección se confirmó usando retrotransposition competente y construcciones incompetentes y sondas para detect SNEO o la neomicina y globina intrón 16. Esto explica la metodología para algunas de las deficiencias de los ensayos de retrotransposición base de cultivo de células, tales como inserciones múltiples, resistencia a la colonia y la expansión clon favorable.

<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.

Un diagrama esquemático del vector retrotransposition L1 se presenta en la Figura 1. El vector consiste en un gen de la neomicina en la orientación antisentido con respecto a L1 ORFs que está interrumpido por un intrón de globina en la orientación sentido y emparedada por SD y SA sitios. Cuando se integra de forma estable en un cromosoma, L1 mRNA se transcribe a partir del CMV y el promotor combinado L1-5&#39;UTR (Figura 1).</stron...

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

Análisis de retrotransposition LINE-1 en el Núcleo Un piso

Aquí empleamos la metodología FISH para realizar un seguimiento de retrotransposition LINE-1 a nivel individual en el cromosoma núcleos diferenciales de líneas de células HepG2 que expresan de forma estable LINE-1 sintético.

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

Método para la medición de la cinética de fusión viral a nivel de partícula

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

Investigación

Biología

Direct Analysis of Single Cells by Mass Spectrometry at Atmospheric Pressure

El análisis directo de las células individuales mediante espectrometría de masas a presión atmosférica

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

El estudio de la proteólisis de ciclina B en el nivel de células individuales en poblaciones de células enteras

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

Agroinfiltration eficiente de las plantas para la expresión transitoria de alto nivel de proteínas recombinantes

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

Análisis de RNA Global Síntesis en el nivel de células individuales siguiente Hipoxia

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

Protocolo CÚBICO Visualiza la expresión de la proteína en la resolución de la celda individual en su totalidad para la piel Mount

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

Análisis de la función de célula Beta usando proyección de imagen de resolución sola célula calcio en islotes de pez cebra

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

Visualizar la interacción entre la proteína marcada Qdot y λ Site-specifically modificada de ADN en el nivel de molécula única

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

Sondeo de mRNA cinética en el espacio y el tiempo en Escherichia coli usando hibridación de fluorescencia de una sola molécula de dos colores

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