El original GFP-Pem1 fue un regalo del doctor Li Lei. Este trabajo fue apoyado por subvenciones del NIH y la Ellison Medical Foundation de VG y AS

En este protocolo se describe el método para el análisis de la reparación del ADN con el reportero del OSD integrado cromosómicamente construcciones de 1,2 en DSBs son inducidos en vivo por la expresión transitoria una endonucleasa de corte rara I-SCE 3. El ensayo integrado proporciona la ventaja de analizar la reparación del OSD en el contexto de los cromosomas. Sin embargo, este protocolo requiere células prolongada pases, lo que puede ser problemático cuando se trabaja con células primarias. Un enfoque alternativo es un ensayo extracromosómico en reportero construcciones se digieren in vitro con el I-SCE o endonucleasas HindIII y transfectadas en las células como ADN lineal. El protocolo del ensayo exctrachromosomal 4 es similar a la prueba integrada se describe a continuación con las siguientes modificaciones. Para el ensayo de extracromosómico omitir la integración de las construcciones de reportero y en su lugar co-transfectar las células con 0,5 ug de reportero NHEJ plásmido linealizado in vitro con el I-SCE o HindIII y 0,1 mg de pDsRed2-N1 como control de transfección. Para el ensayo de recursos humanos co-transfectar 2 reportero HR mg plásmido linearizado con I-SCE o HindIII y 0,1 mg pDsRed2-N1. Analizar las células por FACS tres días después de la transfección. El ensayo extracromosómico permite el análisis de la reparación del OSD en los aislados primarios, evitando celular extensa pases, y facilitar el análisis de múltiples líneas celulares. 1. Construye reportero de NHEJ y de recursos humanos El cassette reportero NHEJ 5 contiene un gen GFP con una ingeniería intrón 3 kb del gen Pem1 (GFP-Pem1). El intrón Pem1 contiene un exón adenoviral flanqueado por secuencias de reconocimiento para las endonucleasas HindIII y SCE (Figura 1a) para la inducción de DSBs. Las páginas de I-SCE se encuentran en orientación invertida. I-SCE tiene una secuencia de reconocimiento nonpalindromic, por lo tanto, dos sitios invertido generar extremos incompatibles ADN (Figura 1c). ADN incompatible termina mejor imitar el DSBs natural. El casete intacta NHEJ es GFP negativas como el exón adenoviral interrumpe la ORF GFP. Tras la inducción de DSBs por I-II CPE, el exón adenoviral se retira y NHEJ restaura la función del gen de la GFP (Figura 2). La característica única de este reportero NHEJ es que se puede detectar una amplia gama de eventos NHEJ desde el intrón puede tolerar eliminaciones e inserciones. El cassette reportero HR 1 (Figura 1b) también se basa en la GFP-Pem1. En la cinta de recursos humanos, el primer exón de la GFP-Pem1 contiene una deleción de 22 pb en combinación con la inserción de tres sitios de restricción, I-SceI/HindIII/I-SceI. La supresión se asegura de que las buenas prácticas agrarias no se puede reconstituir un evento NHEJ. Los dos me-SCE sitios se encuentran en orientación invertida, de manera que I-SCE digestión hojas fines incompatibles (Figura 1c). La primera copia de la GFP-Pem1 es seguido por un promoter-less/ATG-less primer exón y el intrón de GFP-Pem1. La construcción está intacta GFP-negativos. Tras la inducción de un OSD por I-SCE digestión del gen GFP funcional se reconstituye por la conversión de genes intramolecular o intermolecular entre las dos copias mutadas de la primera GFP-Pem1 exón. Desde la segunda copia del gen de la GFP se carece del codón ATG primero y el segundo exón, cruzar o recocido de cadena sencilla no va a restaurar la actividad de las buenas prácticas agrarias. Este diseño permite la detección exclusiva de la conversión de genes, que es la vía de recursos humanos predominante en células de mamíferos (Figura 3). 2. Integración de los reportero construye en el genoma <ol><li> Hacer una preparación de alta calidad de NHEJ o reportero de recursos humanos del plásmido. Para obtener los mejores resultados, utilice Qiagen Endo Kit gratis. Medir la concentración de plásmido y la pureza de la absorbancia a 260/280 nm. </li><li> Linealizar 10 mg del plásmido reportero por digestión con 50 U de NheI en 50 l (volumen total de la reacción) durante 6 horas a 37 ° C incubadora (no utilice el bloque seco, para evitar la evaporación). Purificar el ADN de la solución de digestión con Qiagen QiaexII Kit, eluir el ADN en 20 l de 10 mM Tris-HCl pH 8,0. Medida de concentración de ADN y la pureza de la absorbancia a 260/280 nm. Por lo general, se pierde un 20-30% del ADN durante la purificación. Confirmar el rendimiento y la digestión mediante la ejecución de una pequeña alícuota (2 l) en un gel, junto con un reportero de la construcción de digerir. La construcción purificada reportero lineal se pueden almacenar a 20 ° C. </li><li> En preparación para la transfección, las células de traer a las mejores condiciones de crecimiento. Si a partir de un vial congelado, dividir las células dos veces antes de la transfección. Si a partir de una placa de células confluentes, dividir una vez. </li><li> Crecer las células a un 70-80% de confluencia (por lo general dos días, si plateado 5x10 5 mm células/100 placa, por los fibroblastos humanos normales). Es fundamental para la mejor eficiencia de la transfección de llevar las células en crecimiento logarítmico. La cultura en crecimiento activo contiene células en fase M (visto como células redondeadas unidas a la superficie). </li><li> Transfect las células con las recomendaciones del fabricante Amaxa siguientes Nucleofector es. Para utilizar la solución de los fibroblastos NHDF y Sub-20 del programa. Para la transfección, las células de la cosecha a partir de dos placas de 100 mm (~ 2x10 6 células). Es muy importante que no trypsinize sobre las células. Dejar de tripsinización tan pronto como el 80% de células separadas de la placa. Resuspender las células en una solución de NHDF. Las células son sensibles a la solución de NHDF, por lo tanto, minimizar el tiempo de incubación y mezclar suavemente las células. Transfectar las células con 0,5 ug de reportero construir en forma lineal. Estas condiciones de transfección, cuando se utiliza con el resultado de los fibroblastos humanos normales en la integración de una sola copia de un reportero de la construcción de la mayoría de los integrantes. </li><li> Seleccione las celdas con las construcciones de reportero cromosómicamente integrado por la adición de 1 mg / ml de geneticina (G418) 24 horas después de la transfección. Continuar la selección durante 7-10 días. </li><li> Elige las colonias resistentes a G418 (usted puede escoger las colonias individuales o en la piscina de los clones resistentes). Ampliar la cultura de aproximadamente cinco placas de 100 mm. Congelación de tres platos para su uso futuro y ampliar otros dos placas con el número de células deseadas (por lo general diez placas de 100 mm son suficientes para cinco transfecciones). </li><li> Cuando las células alcanzan 70-80% de confluencia (dos días después de placas 5x10 5 células por placa de 100 mm para los fibroblastos), las células están listos para ser transfectadas con I-II CPE expresión del plásmido. </li></ol> 3. La inducción de la OSD por la expresión transitoria de la I-SCE endonucleasa <ol><li> Transfectar ~ 2x10 6 células (dos placas) de la cultura logarítmica de crecimiento con una mezcla de 5 g I-SCE expresar plásmido y 0,1 mg pDsRed2-N1 (Clontech) como un control de eficiencia de transfección utilizando Amaxa Nucleofector (utilizar la solución de NHDF y el programa Sub-20 de los fibroblastos ). </li><li> Al mismo tiempo, preparar tres controles de calibración para FACS por trasfecting ~ 2x10 6 células con (i) 5 mg EGFP-expresando plásmido, (ii) 5 mg pDsRed2-N1, (iii) 5 g de un plásmido de control que no expresan una la proteína fluorescente (estamos utilizando un plásmido que codifica minigene HPRT). </li><li> Crecer las células durante cuatro días para dar tiempo suficiente para la expresión de la GFP y DsRed proteínas. </li><li> (Opcional) Tres días después de la transfección verificar la eficacia de la transfección y expresión de las buenas prácticas agrarias y de las proteínas fluorescentes DsRed por microscopio invertido con filtros para la detección de buenas prácticas agrarias y DsRed. </li></ol> 4. Análisis de la GFP + y células DsRed + por citometría de flujo <ol><li> Cuatro días después de la transfección de la cosecha I-SCE células transfectadas y el control de las células. Resuspender las células en 500 l de PBS y la transferencia de células a las trompas de FACS. En esta etapa es fundamental para cosechar todas las células y tienen células de forma redonda. Para lograr esto, se recomienda utilizar un tiempo más largo o un tripsinización tripsina más fuerte. Confirmar que el 99% de las células se separan de la placa y tienen forma redonda al observar las células bajo el microscopio. Mantener las células en hielo protegido de la luz (cubierta cubo de hielo con papel de aluminio). Es posible almacenar las células en hielo durante unas horas. Para obtener los mejores resultados de análisis de las células inmediatamente después de la cosecha. </li><li> Calibrar FACS con las buenas prácticas agrarias, DsRed y los controles negativos. Ajustar el voltaje y la compensación de color con el fin de incluir todas las células fluorescentes en el análisis (Figura 4). Tenga en cuenta que la intensidad de fluorescencia de las muestras experimentales será más baja que la de los controles. </li><li> Analizar I-SCE células transfectadas por FACS. Contamos con al menos 20.000 células por cada tratamiento. Para evitar posibles interferencias entre las buenas prácticas agrarias y DsRed fluorescencia se recomienda para mantener el porcentaje de las buenas prácticas agrarias + o DsRed + células por debajo del 25%. Si el porcentaje de DsRed + o células GFP + es más alto reducirá la cantidad de pDsRed2-N1 o me SCE-plásmido. En nuestros experimentos sólo se necesita para ajustar la cantidad de pDsRed2-N1 plásmido, pero no del plásmido I-SCE. </li><li> El porcentaje de células GFP + corresponde a la eficiencia de reparación del ADN del OSD y el porcentaje de células + DsRed indica la eficiencia de la transfección. Calcular la eficiencia relativa de reparación del ADN OSD como la proporción de células GFP + + en las células DsRed. </li><li> Las uniones final reparados pueden ser secuenciados para probar la exactitud de la reparación. El reportero construcciones contienen el origen de la replicación de E. coli y los genes de resistencia a la kanamicina (Figura 1). Esto permite el rescate de las construcciones por la digestión del ADN genómico con EcoRI enzima, recircularization, y su transformación en células competentes de E. coli. Los plásmidos rescatados son analizadas por la digestión de restricción y secuenciación. </li></ol> 5. Resultados representante FACS resultados típicos de la transfección de control y de NHEJ y experimentos de recursos humanos en las Figuras 4 y 5. La intensidad de fluorescencia y el número de células fluorescentes serán más bajos en las células I-SCE transfectadas que contienen integrared NHEJ y construye HR (Figura 5) en comparación con los controles (Figura 4) debido a la diferencia en el número de copias de las buenas prácticas agrarias y DsRed construye en estas células. Cada célula en el experimento sólo contiene una copia del reportero construir integrada, los eventos de reparación tanto éxito se reconstituirá el gen de la GFP en una fracción de las células. Transfección con 0,1 mg pDsRed2-N1 en fibroblastos humanos ofrece aproximadamente una copia del plásmido por célula. La eficiencia de los recursos humanos y NHEJ se calcula como la relación de las buenas prácticas agrarias + / DsRed + en las células. NHEJ es un proceso más eficiente que la de recursos humanos en las células humanas 2. En los fibroblastos humanos de la eficiencia NHEJ es típicamente 0.6-1.3, y la eficiencia de recursos humanos es 0.05-0.3. Dado que la eficiencia del OSD se mide como una proporción de GFP + / DsRed + variaciones de las células de la mezcla de I-SCE y los plásmidos DsRed2-N1 puede afectar al resultado. La calidad del plásmido también puede afectar los resultados al cambiar la eficiencia de transfección. Por lo tanto, para obtener medidas consistentes, es importante utilizar la mezcla mismo plásmido dentro de un experimento. Por otra parte, la eficiencia de reparación del OSD se ve afectada por el tipo de células, la fase del ciclo celular y localización cromosómica de las construcciones integradas. <img src="/files/ftp_upload/2002/2002fig1.jpg" alt="Figura 1" /> Figura 1. Reportero construye para el análisis de la reparación del ADN OSD por NHEJ (A) y HR (B). (C) del ADN incompatibles extremos generados por la digestión de dos invertida I-SCE sitios SD, empalme de donantes, SA, aceptor de empalme, sombreadas plazas, sitios de poliadenilación, Neo / Kana, único ORF controlada por dos promotores:. SV40 que confiere resistencia a la neomicina en los mamíferos las células, y β-lactamasa que confiere resistencia kanamicyn en E. coli, Ori, E. Coli pUC origen de replicación. <img src="/files/ftp_upload/2002/2002fig2.jpg" alt="Figura 2" /> Figura 2. Reportero construir para el análisis de NHEJ En esta construcción el gen de la GFP está inactivo;. Sin embargo, la inducción de un NHEJ OSD y el éxito se convierte en la construcción GFP +. A continuación se muestra un producto de reparación de este reportero por NHEJ. <img src="/files/ftp_upload/2002/2002fig3.jpg" alt="Figura 3" /> Figura 3. Reportero construir para el análisis de recursos humanos por la conversión de genes. Tras la inducción de la DSBs por I-SCE, de recursos humanos por conversión génica (GC) reconstituye un gen GFP activo. A continuación se muestran los productos de reparación de este reportero por la vías de reparación del ADN importantes: GC, cruzando (X-over), solo capítulo recocido (SSA), y NHEJ. X-over reparación del producto se muestra para la recombinación intramolecular, la recombinación exramolecular dará el mismo producto pero se encuentra en un cromosoma. Los genes que expresan proteínas GFP activa (GFP +) se indican con color verde. <img src="/files/ftp_upload/2002/2002fig4.jpg" alt="Figura 4" /> Figura 4. Calibración de los parámetros para el análisis de FACS. (A) Los fibroblastos transfectados con el plásmido pHPRT, utilizado como control negativo para excluir a las células auto fluorescentes. (B) Los fibroblastos transfectados con el plásmido pEGFP, que se utiliza para ajustar la puerta de enlace para las buenas prácticas agrarias + células (zona verde). (C) Los fibroblastos transfectados con el plásmido pDsRed2-N1, que se utiliza para ajustar la puerta de enlace para las células + DsRed (zona roja). <img src="/files/ftp_upload/2002/2002fig5.jpg" alt="Figura 5" /> Figura 5. Los resultados típicos del análisis de NHEJ (A) y HR (B) en los fibroblastos humanos normales con las construcciones de reportero integrado cromosómicamente.

<ol>
	<li>Mao, Z., Seluanov, A., Jiang, Y., Gorbunova, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TRF2+is+required+for+repair+of+nontelomeric+DNA+double-strand+breaks+by+homologous+recombination.">TRF2 is required for repair of nontelomeric DNA double-strand breaks by homologous recombination.</a> Proc Natl Acad Sci U S A. 104, 13068-13073 (2007).</li><li>Mao, Z., Bozzella, M., Seluanov, A., Gorbunova, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+nonhomologous+end+joining+and+homologous+recombination+in+human+cells.">Comparison of nonhomologous end joining and homologous recombination in human cells.</a> DNA Repair (Amst). 7, 1765-1771 (2008).</li><li>Rouet, P., Smih, F., Jasin, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+of+a+site-specific+endonuclease+stimulates+homologous+recombination+in+mammalian+cells.">Expression of a site-specific endonuclease stimulates homologous recombination in mammalian cells.</a> Proc Natl Acad Sci U S A. 91, 6064-6068 (1994).</li><li>Mao, Z., Jiang, Y., Xiang, L., Seluanov, A., Gorbunova, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+repair+by+homologous+recombination,+but+not+by+nonhomologous+end+joining,+is+elevated+in+breast+cancer+cells.">DNA repair by homologous recombination, but not by nonhomologous end joining, is elevated in breast cancer cells.</a> Neoplasia. 11, 683-691 (2009).</li><li>Seluanov, A., Mittelman, D., Pereira-Smith, O. M., Wilson, J. H., Gorbunova, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+end+joining+becomes+less+efficient+and+more+error-prone+during+cellular+senescence.">DNA end joining becomes less efficient and more error-prone during cellular senescence.</a> Proc Natl Acad Sci U S A. 101, 7624-7629 (2004).</li><li>Mao, Z., Bozzella, M., Seluanov, A., Gorbunova, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+repair+by+nonhomologous+end+joining+and+homologous+recombination+during+cell+cycle+in+human+cells.">DNA repair by nonhomologous end joining and homologous recombination during cell cycle in human cells.</a> Cell Cycle. 7, 2902-296 (2008).</li></ol>

Los fluorescentes NHEJ y ensayos HR reportero proporcionan una forma cuantitativa para medir por separado cada vía de reparación del OSD en vivo. Los ensayos son muy sensibles, como FACS puede detectar con fiabilidad 10 células GFP + de 20.000 células. Los ensayos pueden ser adaptados para medir los eventos de reparación en &quot;tiempo real&quot; mediante la detección de la aparición de células GFP + en minutos u horas después de la inducción de DSBs 2. Además, el análisis de las célula...

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		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
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<td>EndoFree Plasmid Maxi kit</td>
<td>&nbsp;</td>
<td>Qiagen</td>
<td>12362</td>
<td>&nbsp;</td>
<tr>
<td>Qiaex II Gel Extraction Kit</td>
<td>&nbsp;</td>
<td>Qiagen</td>
<td>20021</td>
<td>&nbsp;</td>
<tr>
<td>Amaxa Nucleofector</td>
<td>&nbsp;</td>
<td>Lonza</td>
<td>AAD-1001</td>
<td>&nbsp;</td>
<tr>
<td>Geneticin (G418)</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>11811-031</td>
<td>&nbsp;</td>
<tr>
<td>pDsRed2-N1</td>
<td>&nbsp;</td>
<td>Clontech</td>
<td>632406</td>
<td>&nbsp;</td>
<tr>
<td>Round bottom tubes</td>
<td>&nbsp;</td>
<td>BD Falcon</td>
<td>352058</td>
<td>FACS tubes</td>
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analysis of dna double-strand break (dsb) repair in mammalian cells

ADN de doble filamento se rompe son las lesiones del ADN más peligrosa que puede conducir a la pérdida masiva de información genética y la muerte celular. Las células de reparación de DSBs con dos vías principales: no homólogos fin de unirse (NHEJ) y recombinación homóloga (HR). Las perturbaciones de NHEJ y de recursos humanos a menudo se asocian con el envejecimiento prematuro y la tumorogénesis, por lo tanto es importante tener una forma cuantitativa de medición de cada vía de reparación del OSD. Nuestro laboratorio ha desarrollado estructuras fluorescentes reportero que permiten la medición sensible y cuantitativo de NHEJ y de recursos humanos. Las construcciones se basan en un gen GFP diseñado que contiene sitios de reconocimiento para un poco de corte I-II CPE endonucleasa para la inducción de DSBs. Las construcciones de partida son las buenas prácticas agrarias negativas como el gen de la GFP se inactiva por un exón adicional, o por mutaciones. Éxito de la reparación de la I-SCE-inducida por NHEJ rompe o HR restaura el gen GFP funcional. El número de células GFP positivos contados por citometría de flujo proporciona una medida cuantitativa de NHEJ o la eficiencia de recursos humanos.

Watch this Scientific Journal Video about Analysis of DNA Double-strand Break DSB Repair in Mammalian Cells at JoVE.com

Analysis of DNA Double-strand Break DSB Repair in Mammalian Cells

En este artículo se describen las buenas prácticas agrarias basadas en la fluorescencia<em&gt; In vivo</em&gt; Pruebas que cuantificar por separado la recombinación homóloga y no homóloga fin de unirse en células de mamíferos.

El análisis de ADN de doble cadena Break (OSD) de reparación en células de mamífero

DNA double-strand breaks are the most dangerous DNA lesions that may lead to massive loss of genetic information and cell death.  Cells repair DSBs using ...

analysis-of-dna-double-strand-break-dsb-repair-in-mammalian-cells

Research

JoVE Journal

Biology

Analysis of DNA Double-strand Break (DSB) Repair in Mammalian Cells

31.5K vistas.  University of Rochester. En este artículo se describen las buenas prácticas agrarias basadas en la fluorescencia In vivo Pruebas que cuantificar por separado la recombinación homóloga y no homóloga fin de unirse en células de mamíferos.

Video: Analysis of DNA Double-strand Break DSB Repair in Mammalian Cells

DNA Transfection of Mammalian Skeletal Muscles using In Vivo Electroporation

A growing interest in cell biology is to express transgenically modified forms of essential proteins (e.g. fluorescently tagged constructs and/or mutant variants) in order to investigate their endogenous distribution and functional relevance. An interesting approach that has been implemented to fulfill this objective in fully differentiated cells is the in vivo transfection of plasmids by various methods into specific tissues such as liver1, skeletal muscle2,3, and even the brain4. We present here a detailed description of the steps that must be followed in order to efficiently transfect genetic material into fibers of the flexor digitorum brevis (FDB) and interosseus (IO) muscles of adult mice using an in vivo electroporation approach. The experimental parameters have been optimized so as to maximize the number of muscle fibers transfected while minimizing tissue damages that may impair the quality and quantity of the proteins expressed in individual fibers. We have verified that the implementation of the methodology described in this paper results in a high yield of soluble proteins, i.e. EGFP and ECFP3, calpain, FKBP12, &beta;2a-DHPR, etc. ; structural proteins, i.e. minidystrophin and &alpha;-actinin; and membrane proteins, i.e. &alpha;1s-DHPR, RyR1, cardiac Na/Ca2+ exchanger , NaV1.4 Na channel, SERCA1, etc., when applied to FDB, IO and other muscles of mice and rats. The efficient expression of some of these proteins has been verified with biochemical3 and functional evidence5. However, by far the most common confirmatory approach used by us are standard fluorescent microscopy and 2-photon laser scanning microscopy (TPLSM), which permit to identify not only the overall expression, but also the detailed intracellular localization, of fluorescently tagged protein constructs. The method could be equally used to transfect plasmids encoding for the expression of proteins of physiological relevance (as shown here), or for interference RNA (siRNA) aiming to suppress the expression of normally expressed proteins (not tested by us yet). It should be noted that the transfection of FDB and IO muscle fibers is particularly relevant for the investigation of mammalian muscle physiology since fibers enzymatically dissociated from these muscles are currently one of the most suitable models to investigate basic mechanisms of excitability and excitation-contraction coupling under current or voltage clamp conditions2,6-8.

DNA Transfection of Mammalian Skeletal Muscles using In Vivo Electroporation

We describe detailed procedures for the efficient transfection of plasmid DNA into the fibers of foot muscles of live mice using electroporation and the subsequent visualization of protein expression using fluorescence microscopy.

La transfección de ADN de músculos esqueléticos de mamíferos con En Vivo La electroporación

 A growing interest in cell biology is to express transgenically modified forms of essential proteins (e.g. fluorescently tagged constructs and/or mutant ...

Investigación

Biología

Intranuclear Microinjection of DNA into Dissociated Adult Mammalian Neurons

Primary neuronal cell cultures are valuable tools to study protein function since they represent a more biologically relevant system compared to immortalized cell lines. However, the post-mitotic nature of primary neurons prevents effective heterologous protein expression using common procedures such as electroporation or chemically-mediated transfection. Thus, other techniques must be employed in order to effectively express proteins in these non-dividing cells.
In this article, we describe the steps required to perform intranuclear injections of cDNA constructs into dissociated adult sympathetic neurons. This technique, which has been applied to different types of neurons, can successfully induce heterologous protein expression. The equipment essential for the microinjection procedure includes an inverted microscope to visualize cells, a glass injection pipet filled with cDNA solution that is connected to a N2(g) pressure delivery system, and a micromanipulator. The micromanipulator coordinates the injection motion of microinjection pipet with a brief pulse of pressurized N2 to eject cDNA solution from the pipet tip. 
This technique does not have the toxicity associated with many other transfection methods and enables multiple DNA constructs to be expressed at a consistent ratio. The low number of injected cells makes the microinjection procedure well suited for single cell studies such as electrophysiological recordings and optical imaging, but may not be ideal for biochemical assays that require a larger number of cells and higher transfection efficiencies. Although intranuclear microinjections require an investment of equipment and time, the ability to achieve high levels of heterologous protein expression in a physiologically relevant environment makes this technique a very useful tool to investigate protein function.

Direct intranuclear injection of cDNA is an effective transfection technique for post-mitotic cells. This method provides high levels of heterologous protein expression from single or multiple cDNA constructs and enables protein function to be studied in a physiologically relevant environment with a variety of single cell assays.

La microinyección de ADN en intranucleares disociada neuronas de mamíferos adultos

Primary neuronal cell cultures are valuable tools to study protein function since they represent a more biologically relevant system compared to ...

Pulse-chase Analysis of N-linked Sugar Chains from Glycoproteins in Mammalian Cells

Attachment of the Glc3Man9GlcNAc2 precursor oligosaccharide to nascent polypeptides in the ER is a common modification for secretory proteins. Although this modification was implicated in several biological processes, additional aspects of its function are emerging, with recent evidence of its role in the production of signals for glycoprotein quality control and trafficking. Thus, phenomena related to N-linked glycans and their processing are being intensively investigated. Methods that have been recently developed for proteomic analysis have greatly improved the characterization of glycoprotein N-linked glycans. Nevertheless, they do not provide insight into the dynamics of the sugar chain processing involved. For this, labeling and pulse-chase analysis protocols are used that are usually complex and give very low yields. We describe here a simple method for the isolation and analysis of metabolically labeled N-linked oligosaccharides. The protocol is based on labeling of cells with [2-3H] mannose, denaturing lysis and enzymatic release of the oligosaccharides from either a specifically immunoprecipitated protein of interest or from the general glycoprotein pool by sequential treatments with endo H and N-glycosidase F, followed by molecular filtration (Amicon). In this method the isolated oligosaccharides serve as an input for HPLC analysis, which allows discrimination between various glycan structures according to the number of monosaccharide units comprising them, with a resolution of a single monosaccharide. Using this method we were able to study high mannose N-linked oligosaccharide profiles of total cell glycoproteins after pulse-chase in normal conditions and under proteasome inhibition. These profiles were compared to those obtained from an immunoprecipitated ER-associated degradation (ERAD) substrate. Our results suggest that most NIH 3T3 cellular glycoproteins are relatively stable and that most of their oligosaccharides are trimmed to Man9-8GlcNAc2. In contrast, unstable ERAD substrates are trimmed to Man6-5GlcNAc2 and glycoproteins bearing these species accumulate upon inhibition of proteasomal degradation.

We describe a method for analysis of the alteration of N-linked glycans through the early life of glycoproteins after their biosynthesis in mammalian cells. This is achieved by pulse-chase analysis of metabolically labeled glycans, enzymatic release from glycoproteins and examination by HPLC.

Pulso-caza de análisis de cadenas de azúcares unidos a N de glicoproteínas en células de mamífero

Attachment of the Glc3Man9GlcNAc2 precursor oligosaccharide to nascent polypeptides in the ER is a common modification for secretory proteins. Although ...

Mutagenesis and Functional Analysis of Ion Channels Heterologously Expressed in Mammalian Cells

We will demonstrate how to study the functional effects of introducing a point mutation in an ion channel. We study G protein-gated inwardly rectifying potassium (referred to as GIRK) channels, which are important for regulating the excitability of neurons. There are four different mammalian GIRK channel subunits (GIRK1-GIRK4) - we focus on GIRK2 because it forms a homotetramer. Stimulation of different types of G protein-coupled receptors (GPCRs), such as the muscarinic receptor (M2R), leads to activation of GIRK channels. Alcohol also directly activates GIRK channels. We will show how to mutate one amino acid by specifically changing one or more nucleotides in the cDNA for the GIRK channel. This mutated cDNA sequence will be amplified in bacteria, purified, and the presence of the point mutation will be confirmed by DNA sequencing. The cDNAs for the mutated and wild-type GIRK channels will be transfected into human embryonic kidney HEK293T cells cultured in vitro. Lastly, whole-cell patch-clamp electrophysiology will be used to study the macroscopic potassium currents through the ectopically expressed wild-type or mutated GIRK channels. In this experiment, we will examine the effect of a L257W mutation in GIRK2 channels on M2R-dependent and alcohol-dependent activation.

We will demonstrate how to study the effect of a single point mutation on the function of an ion channel.

Mutagénesis y análisis funcional de los canales iónicos heteróloga se expresa en células de mamífero

We will demonstrate how to study the functional effects of introducing a point mutation in an ion channel. We study G protein-gated inwardly rectifying ...

Analysis of Schwann-astrocyte Interactions Using In Vitro Assays

Schwann cells are one of the commonly used cells in repair strategies following spinal cord injuries. Schwann cells are capable of supporting axonal regeneration and sprouting by secreting growth factors 1,2 and providing growth promoting adhesion molecules 3 and extracellular matrix molecules 4. In addition they myelinate the demyelinated axons at the site of injury 5.
However following transplantation, Schwann cells do not migrate from the site of implant and do not intermingle with the host astrocytes 6,7. This results in formation of a sharp boundary between the Schwann cells and astrocytes, creating an obstacle for growing axons trying to exit the graft back into the host tissue proximally and distally. Astrocytes in contact with Schwann cells also undergo hypertrophy and up-regulate the inhibitory molecules 8-13.
In vitro assays have been used to model Schwann cell-astrocyte interactions and have been important in understanding the mechanism underlying the cellular behaviour.
These in vitro assays include boundary assay, where a co-culture is made using two different cells with each cell type occupying different territories with only a small gap separating the two cell fronts. As the cells divide and migrate, the two cellular fronts get closer to each other and finally collide. This allows the behaviour of the two cellular populations to be analyzed at the boundary. Another variation of the same technique is to mix the two cellular populations in culture and over time the two cell types segregate with Schwann cells clumped together as islands in between astrocytes together creating multiple Schwann-astrocyte boundaries.
The second assay used in studying the interaction of two cell types is the migration assay where cellular movement can be tracked on the surface of the other cell type monolayer 14,15. This assay is commonly known as inverted coverslip assay. Schwann cells are cultured on small glass fragments and they are inverted face down onto the surface of astrocyte monolayers and migration is assessed from the edge of coverslip.
Both assays have been instrumental in studying the underlying mechanisms involved in the cellular exclusion and boundary formation. Some of the molecules identified using these techniques include N-Cadherins 15, Chondroitin Sulphate proteoglycans(CSPGs) 16,17, FGF/Heparin 18, Eph/Ephrins19.
This article intends to describe boundary assay and migration assay in stepwise fashion and elucidate the possible technical problems that might occur.

Analysis of Schwann-astrocyte Interactions Using In Vitro Assays

This article intends to describe in stepwise fashion the commonly used in vitro assays used in studying Schwann cell-asrtocyte interactions.

El análisis de Schwann-astrocito interacciones utilizando In Vitro Ensayos

Schwann cells are one of the commonly used cells in repair strategies following spinal cord injuries. Schwann cells are capable of supporting axonal ...

Analysis of mRNA Nuclear Export Kinetics in Mammalian Cells by Microinjection

In eukaryotes, messenger RNA (mRNA) is transcribed in the nucleus and must be exported into the cytoplasm to access the translation machinery. Although the nuclear export of mRNA has been studied extensively in Xenopus oocytes1 and genetically tractable organisms such as yeast2 and the Drosophila derived S2 cell line3, few studies had been conducted in mammalian cells. Furthermore the kinetics of mRNA export in mammalian somatic cells could only be inferred indirectly4,5. In order to measure the nuclear export kinetics of mRNA in mammalian tissue culture cells, we have developed an assay that employs the power of microinjection coupled with fluorescent in situ hybridization (FISH). These assays have been used to demonstrate that in mammalian cells, the majority of mRNAs are exported in a splicing dependent manner6,7, or in manner that requires specific RNA sequences such as the signal sequence coding region (SSCR) 6. In this assay, cells are microinjected with either in vitro synthesized mRNA or plasmid DNA containing the gene of interest. The microinjected cells are incubated for various time points then fixed and the sub-cellular localization of RNA is assessed using FISH. In contrast to transfection, where transcription occurs several hours after the addition of nucleic acids, microinjection of DNA or mRNA allows for rapid expression and allows for the generation of precise kinetic data.

Here we describe an assay that employs the power of microinjection coupled with fluorescent in situ hybridization in order to accurately measure the nuclear export kinetics of mRNA in mammalian somatic cells.

Análisis de la cinética de exportación nuclear de ARNm en células de mamífero por microinyección

In eukaryotes, messenger RNA (mRNA) is transcribed in the nucleus and must be exported into the cytoplasm to access the translation machinery. Although ...

Analysis of Cell Cycle Position in Mammalian Cells

The regulation of cell proliferation is central to tissue morphogenesis during the development of multicellular organisms. Furthermore, loss of control of cell proliferation underlies the pathology of diseases like cancer. As such there is great need to be able to investigate cell proliferation and quantitate the proportion of cells in each phase of the cell cycle. It is also of vital importance to indistinguishably identify cells that are replicating their DNA within a larger population. Since a cell&prime;s decision to proliferate is made in the G1 phase immediately before initiating DNA synthesis and progressing through the rest of the cell cycle, detection of DNA synthesis at this stage allows for an unambiguous determination of the status of growth regulation in cell culture experiments.
DNA content in cells can be readily quantitated by flow cytometry of cells stained with propidium iodide, a fluorescent DNA intercalating dye. Similarly, active DNA synthesis can be quantitated by culturing cells in the presence of radioactive thymidine, harvesting the cells, and measuring the incorporation of radioactivity into an acid insoluble fraction. We have considerable expertise with cell cycle analysis and recommend a different approach. We Investigate cell proliferation using bromodeoxyuridine/fluorodeoxyuridine (abbreviated simply as BrdU) staining that detects the incorporation of these thymine analogs into recently synthesized DNA. Labeling and staining cells with BrdU, combined with total DNA staining by propidium iodide and analysis by flow cytometry1 offers the most accurate measure of cells in the various stages of the cell cycle. It is our preferred method because it combines the detection of active DNA synthesis, through antibody based staining of BrdU, with total DNA content from propidium iodide. This allows for the clear separation of cells in G1 from early S phase, or late S phase from G2/M. Furthermore, this approach can be utilized to investigate the effects of many different cell stimuli and pharmacologic agents on the regulation of progression through these different cell cycle phases.
In this report we describe methods for labeling and staining cultured cells, as well as their analysis by flow cytometry. We also include experimental examples of how this method can be used to measure the effects of growth inhibiting signals from cytokines such as TGF-&beta;1, and proliferative inhibitors such as the cyclin dependent kinase inhibitor, p27KIP1. We also include an alternate protocol that allows for the analysis of cell cycle position in a sub-population of cells within a larger culture5. In this case, we demonstrate how to detect a cell cycle arrest in cells transfected with the retinoblastoma gene even when greatly outnumbered by untransfected cells in the same culture. These examples illustrate the many ways that DNA staining and flow cytometry can be utilized and adapted to investigate fundamental questions of mammalian cell cycle control.

Determining the cell cycle position of a population of cells, or understanding how signals affect proliferation, can be readily measured by flow cytometry using this protocol. We report a simple experimental approach to staining cells and quantifying their position in the cell cycle.

Análisis de la posición del ciclo celular en células de mamífero

The regulation of cell proliferation is central to tissue morphogenesis during the development of multicellular organisms. Furthermore, loss of control of ...

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.

Imágenes en tiempo real de Soltero Engineered ARN transcritos en células vivas utilizando Ratiometric bimoleculares Balizas

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

Measurement of Heme Synthesis Levels in Mammalian Cells

Heme serves as the prosthetic group for a wide variety of proteins known as hemoproteins, such as hemoglobin, myoglobin and cytochromes. It is involved in various molecular and cellular processes such as gene transcription, translation, cell differentiation and cell proliferation. The biosynthesis levels of heme vary across different tissues and cell types and is altered in diseased conditions such as anemia, neuropathy and cancer. This technique uses [4-14C] 5-aminolevulinic acid ([14C] 5-ALA), one of the early precursors in the heme biosynthesis pathway to measure the levels of heme synthesis in mammalian cells. This assay involves incubation of cells with [14C] 5-ALA followed by extraction of heme and measurement of the radioactivity incorporated into heme. This procedure is accurate and quick. This method measures the relative levels of heme biosynthesis rather than the total heme content. To demonstrate the use of this technique the levels of heme biosynthesis were measured in several mammalian cell lines.

Altered intracellular heme levels are associated with common diseases such as cancer. Thus, there is a need to measure heme biosynthesis levels in diverse cells. The goal of this protocol is to provide a fast and sensitive method to measure and compare the levels of heme synthesis in different cells.

Medición de niveles hemo síntesis en células de mamífero

Heme serves as the prosthetic group for a wide variety of proteins known as hemoproteins, such as hemoglobin, myoglobin and cytochromes. It is involved in ...

Examination of Proteins Bound to Nascent DNA in Mammalian Cells Using  BrdU-ChIP-Slot-Western Technique

Histone deacetylases 1 and 2 (HDAC1,2) localize to the sites of DNA replication. In the previous study, using a selective inhibitor and a genetic knockdown system, we showed novel functions for HDAC1,2 in replication fork progression and nascent chromatin maintenance in mammalian cells. Additionally, we used a BrdU-ChIP-Slot-Western technique that combines chromatin immunoprecipitation (ChIP) of bromo-deoxyuridine (BrdU)-labeled DNA with slot blot and Western analyses to quantitatively measure proteins or histone modification associated with nascent DNA.
Actively dividing cells were treated with HDAC1,2 selective inhibitor or transfected with siRNAs against Hdac1 and Hdac2 and then newly synthesized DNA was labeled with the thymidine analog bromodeoxyuridine (BrdU). The BrdU labeling was done at a time point when there was no significant cell cycle arrest or apoptosis due to the loss of HDAC1,2 functions. Following labeling of cells with BrdU, chromatin immunoprecipitation (ChIP) of histone acetylation marks or the chromatin-remodeler was performed with specific antibodies. BrdU-labeled input DNA and the immunoprecipitated (or ChIPed) DNA was then spotted onto a membrane using the slot blot technique and immobilized using UV. The amount of nascent DNA in each slot was then quantitatively assessed using Western analysis with an anti-BrdU antibody. The effect of loss of HDAC1,2 functions on the levels of newly synthesized DNA-associated histone acetylation marks and chromatin remodeler was then determined by normalizing the BrdU-ChIP signal obtained from the treated samples to the control samples.

In this protocol, we describe a novel BrdU-ChIP-Slot-Western technique to examine proteins and histone modifications associated with newly synthesized or nascent DNA.