Este trabajo fue financiado por los Institutos Nacionales de Salud (DP1CA174426 a Carlos F. Barbas) y la Universidad ShanghaiTech, Shanghai, China (a JL). Se generaron gráficos moleculares utilizando PyMOL.

1. Clonación <ol><li> Obtener dominios ZIF con dos dedos de alanina-sustituido que se han clonado sub-en el sistema de vector de expresión pET-28 y están disponibles bajo petición (PET-2F-ZiF). 34 </li><li> Amplificar por PCR EmGFP del plásmido Esmeralda-pBAD con los cebadores 5 &#39;XmaI-EmGFP (5&#39;-GGAAATTG CCCGGG ATGGTGAGCAAGGGCGAGGAGCTGTTCAC-3&#39;; Xma sitio I en negrita) y 3 &#39;Sac-EmGFP (5&#39;-CGGATCT GAGCTC TTACTTGTACAGCTCGTCCATGCCGAG-3&#39; ; Sac I sitio en negrita). <ol><li> Utilice 5 ng de ADN plantilla, 10 l de tampón 10x de la polimerasa, 1 unidades (U) de ADN Taq polimerasa, 0,2 mM de cada dNTP y 0,2 M de cada cebador en una solución de 100 l con el volumen restante compuestos de agua destilada / desionizada . Utilice las condiciones de ciclo: 95 ° C durante 5 minutos; 30 ciclos de 95 ° C durante 30 segundos, 55ºC durante 30 segundos y 72 ° C durante 1 minuto; 72 ° C durante 10 min. Se purifica el producto de PCR por extracti gelen y determinar la concentración de ADN usando un espectrofotómetro de medición Abs 260 x 50 g / ml. </li></ol></li><li> Recopilación pET-2F-ZiF y el inserto que codifica EmGFP con las enzimas de restricción Xma I y Sac I en tampón recomendado durante 3 horas a 37 ° C utilizando 10 U de enzima por 1 g de ADN. Visualizar el ADN por electroforesis en gel de agarosa usando un colorante intercalante fluorescente, tal como bromuro de etidio. </li><li> Se purifica el ADN digerido mediante el kit de extracción de gel y determinar la concentración de ADN mediante un espectrofotómetro de medición Abs 260 x 50 g / ml. </li><li> Ligar el ADN que codifica EmGFP purificado en 50-100 ng de pET-2F-ZiF utilizando 1 U de T4 DNA ligasa durante al menos 1 h a TA. Para obtener los mejores resultados, realice la reacción de ligación utilizando un 6: 1 molar-inserción a vector. </li><li> Descongelar 50 l de cualquier químicamente competentes XL-1 Azul de Escherichia coli células en hielo y mezclar suavemente con 10-20 ng de ligó pET-2F-ZiF-EmGFP. </li><li> Mantenga en hielo durante 30 min. El choque térmico la mezcla a 42 ° C durante 90 seg y recuperar las células en 2 ml de caldo de Super óptima con represión catabólica (SOC) durante 1 hora a 37 ° C con agitación. </li><li> Corre 100 l de la cultura de recuperación en un caldo lisogenia (LB) placa de agar con 50 g / ml de kanamicina y se incuba O / N a 37 ° C. </li><li> El día siguiente, inocular 6 ml de caldo super (SB) o de cultivo LB que contiene 50 mg / ml de kanamicina con una colonia de la placa de agar LB y cultivo O / N a 37 ° C. Nota: Colony PCR usando los cebadores 5 &#39;XmaI-EmGFP y 3&#39; SacI-EmGFP podría utilizarse para identificar los clones positivos antes de la miniprep. </li><li> Purificar pET-2F-ZiF-EmGFP por miniprep y confirmar mediante secuenciación de ADN plásmido utilizando el promotor T7 (5&#39;-TAATACGACTCACTATAGGG-3 &#39;). </li></ol> 2. Expresión y purificación <ol><li> Descongele 50 l de químicamente competente E. BL21 coli células en hielo y mezclar suavemente con 100 ng de pET-2F-ZiF-EmGFP plasmid. Transformar como se describe en los pasos 1.7 a 1.8. </li><li> El día siguiente, inocular 10 ml de medio LB que contenía 50 mg / ml de kanamicina con una sola colonia y crecer O / N a 37 ° C con agitación. </li><li> El día siguiente, diluir 10 ml de cultivo O / N en 1 L de medio LB suplementado con 50 g / ml de kanamicina, 0,2% de glucosa y 100 mM ZnCl2. Mantener el cultivo a 37 ° C con agitación hasta una densidad óptica a 600 nm (OD 600) de 0,8 e inducir la expresión de la proteína con 2 mM isopropil-β-D-1-tiogalactopiranósido (IPTG). Después de 6 horas de crecimiento a 37 ° C, las células de la cosecha por centrifugación a 5000 xg durante 10 min a 4 ° C. Nota: Las condiciones de inducción son altamente variables y dependen de la estabilidad de las proteínas que se expresa. Supervisar el OD 600 cada 30 minutos hasta una DO 600 de 0,8 se alcanza. </li><li> Resuspender el sedimento celular en 5 ml de tampón de lisis (50 mM Tris-HCl, NaCl 500 mM, 100 mM ZnCl2, 1 mM ditiotreitol (DTT), 1 mM de MgCl 2, fluoruro de fenilmetilsulfonilo 1 mM (PMSF) e imidazol 10 mM, pH 8,0). Lisar las células en hielo por ultrasonidos con la siguiente configuración: potencia del 50%, 4 minutos de tiempo de proceso con 5 segundos de encendido / apagado 10 segundos intervalos. Evite el sobrecalentamiento de la solución. </li><li> Centrifugar el lisado de células a 25.000 xg durante 30 min a 4 ° C y transferir el sobrenadante a un tubo de recogida fresco. Para obtener los mejores resultados, realice todos los pasos siguientes a 4 ° C. </li><li> Ejecute el sobrenadante a través de una columna pre-envasados ​​con 1 ml de suspensión equilibrada Ni-NTA. Lavar la resina con 20 ml de tampón de lavado (50 mM Tris-HCl, NaCl 500 mM, 100 mM ZnCl2, DTT 1 mM, 1 mM de MgCl imidazol 2 y 30 mM, pH 8,0). </li><li> Eluir la proteína con 5 ml de tampón de elución (50 mM Tris-HCl, NaCl 500 mM, 100 mM ZnCl2, DTT 1 mM, 1 mM de MgCl 2, e imidazol 300 mM, pH 8,0). </li><li> Intercambio de tampón la proteína se eluyó con tampón de almacenamiento (50 mMTris-HCl, NaCl 500 mM, 100 mM ZnCl2, DTT 1 mM, 1 mM de MgCl 2 y 10% de glicerol, pH 8,0) y concentrar la proteína a al menos 40 mM utilizando un concentrador de giro por centrifugación de acuerdo con las instrucciones del fabricante. </li><li> Determinar la concentración de proteína por el ensayo de Bradford o BCA. Mezclar 2 l de proteínas purificadas con 2 l 2 × colorante de carga SDS-PAGE, hierven a 95 ° C durante 10 min y luego resolver el 4% -20% Tris-glicina SDS-PAGE para evaluar la pureza de proteína (Figura 2). </li></ol> 3. La proteína de almacenamiento <ol><li> Alícuota de la proteína concentrada, congelación de flash en nitrógeno líquido y se almacena a -80 ° C. Para las proteínas de fusión EmGFP, cubrir el tubo con papel de aluminio para proteger la proteína de la luz. </li><li> Evite la congelación y descongelación repetida de la solución de proteína. Nota: Las proteínas fusionadas-ZiF son estables en estas condiciones durante al menos 1 mes. O almacenamiento inadecuado mes&gt; 4 puede conducirpara la precipitación de proteínas o photobleaching de EmGFP. </li></ol> Transducción 4. Proteína <ol><li> Mantener las células HeLa en medio de Eagle modificado por Dulbecco (DMEM) que contiene 10% (v / v) de suero fetal bovino (FBS) y 1% de antibiótico-antimicótico a 37 ° C en una atmósfera totalmente humidificada con 5% de CO 2. Nota: las pasaron más de 30 veces no se recomiendan para la transducción de la proteína. </li><li> Pre-capa de una placa de 24 pocillos con 500 l de 50 mg / ml de poli-lisina durante 30 a 60 min a 25 ° C. Sembrar las células en una placa de 24 pocillos a una densidad de 2 × 10 5 células por pocillo. A las 24 h después de la siembra, o una vez que las células son entre 80% -90% de confluencia, retirar el medio de cada pocillo y se lava con 500 l de DMEM libre de suero pre-calentado (SFM). </li><li> A cada pocillo, añadir 250 l de SFM que contiene 2 M de proteína ZiF-EmGFP y 100 mM ZnCl2. Se incuban las células a 37 ° C durante 1 hr. Nota: Los dominios ZIF entran en las células primarily través macropinocytosis, 34, que es un mecanismo dependiente de la energía. Por lo tanto, las células deben ser incubadas a 37 ° C para la internalización de proteínas eficiente. </li><li> Retire el medio de las células y lavar tres veces con 500 l de calcio y de magnesio libre de solución salina tamponada con fosfato de Dulbecco (DPBS) suplementado con 0,5 mg / ml de heparina. Nota: La heparina es necesario para eliminar la proteína unida a la superficie que pudiera complicar los análisis. </li><li> (Opcional) repetir el tratamiento hasta tres veces para la entrega mejorada. </li><li> Aislar las células tratadas inmediatamente después de la heparina de lavado por digestión con 0,05% de tripsina-EDTA a 37 ° C durante 2 min. Vuelva a suspender las células desprendidas en 0,5 ml de DPBS suplementado con 1% de SFB. </li><li> Medir la intensidad de fluorescencia de cada muestra por citometría de flujo 35 utilizando el canal de isotiocianato de fluoresceína (FITC) (Figura 3). Ajuste la dispersión frontal (FSC) y la dispersión lateral (SSC) para colocar la población deinterés en la escala, asegurando que las poblaciones con diferentes propiedades se resuelven el uno del otro. </li><li> Recoge 10.000 eventos en directo para cada muestra y analizar los datos mediante citometría de flujo análisis de datos de software. 36 Normalizar la fluorescencia de las células HeLa tratadas con ZiF-EmGFP a esas células tratadas sólo con SFM. </li></ol>

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C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure+of+Aart,+a+designed+six-finger+zinc+finger+peptide,+bound+to+DNA.">Structure of Aart, a designed six-finger zinc finger peptide, bound to DNA.</a> J. Mol. Biol. 363, 405-421 (2006).</li></ol>

Los autores no tienen nada que revelar.

Aquí, se presenta un protocolo paso a paso para la entrega de proteínas utilizando zinc-finger (ZiF) dominios de células permeable. El dominio ZiF no reduce la actividad enzimática de la carga fundida 34; permite la producción y purificación de proteínas en rendimientos casi idénticos a los observados con proteína no modificada; y puede transportar proteínas y enzimas en una amplia gama de tipos de células con eficiencias que superan los sistemas de dominio de transducción de proteína o péptido ...

Altamente estrategias de suministro de proteínas eficientes y versátiles son críticas para muchos la investigación básica y las aplicaciones terapéuticas. La entrega directa de proteínas purificadas en las células representa uno de los métodos más seguros y más fáciles para lograr esto. 1,2 diferencia de las estrategias que se basan en la expresión de genes a partir de ácidos nucleicos, 3-5 de administración de proteínas no plantea ningún riesgo de mutagénesis de inserción, es independiente de la maquinaria celular de la transcripción / traducción y permite un efecto inmediato. Sin embargo, la falta de métodos simples y generalizables para dotar a la actividad de penetración celular en proteínas confunde habitualmente su ingreso directo en las células. Los métodos actuales para facilitar la entrega intracelular de proteínas se basan en el uso de origen natural o diseñados 6-8 péptidos de penetración celular, 9-12 dominios de transducción de sobrealimentados, 13,14 nanopartículas y liposomas 15, 16 partículas similares a virus 17,18 </sup&gt; y materiales de microesferas poliméricas. 19 Desafortunadamente, muchos de estos enfoques se ven obstaculizados por las bajas tasas de absorción celular, 20,21 pobre estabilidad, 22 inadvertida especificidad de tipo celular, 23 propiedades de escape bajo endosomales 24 y toxicidad. 25 Además, muchas proteínas tecnologías de transducción de reducir la bioactividad de las proteínas entregados. 14 Nuestro laboratorio previamente demostró que nucleasa dedo de zinc (ZFN) proteínas - restricción endonucleasas quimérico que consiste en una proteína programable Cys2 -Su 2 dedos de zinc de unión a ADN y el dominio de la escisión de la endonucleasa de restricción FokI 26-28 - son inherentemente por células permeable. 29 Esta actividad de penetración celular sorprendente ha demostrado ser una propiedad intrínseca del dominio dedo de zinc de diseño personalizado, una plataforma de unión a ADN que ha surgido como una poderosa herramienta para apuntado genoma eningeniería, 30-32 y considera que es el resultado de la constelación de seis residuos de carga positiva en la superficie de la proteína. De hecho, varias proteínas de unión al ADN, incluyendo c-Jun N-DEK y se han demostrado poseer una capacidad innata para atravesar las membranas celulares. 33 Más recientemente, nuestro laboratorio ampliado en estos resultados y demostró que la actividad de las células de penetración de zinc dedo (ZiF) dominios podrían ser aprovechados para la entrega de proteínas intracelulares. Fusión genética de cualquiera de los dominios ZIF de uno o dos dedos a la carga de proteína específica llevado a la absorción eficiencias que superan muchos sistemas de suministro de péptidos de penetración celular convencionales. 34 En particular, la entrega mediada por ZiF no comprometió la actividad enzimática de la carga fundida y facilitó altos niveles de entrega citosólica. En conjunto, estos resultados demuestran el potencial del dominio ZiF para facilitar la prestación eficiente y fácil de proteínas, y potencialmente más diversos tipos de macromoléculas, en las células. Aquí, se presenta un protocolo detallado paso a paso sobre cómo implementar la tecnología ZiF para la entrega de proteínas en células de mamíferos. Anteriormente hemos construido un conjunto de uno, dos, tres, cuatro, cinco y seis dedo dominios ZIF que carecen de la capacidad de unirse al ADN, debido a la sustitución de cada uno de los residuos de unión a ADN-α helicoidal, pero son capaces de entregar proteínas en células 34 (Figura 1). Se describe la producción y la transducción de Emerald GFP (EmGFP) en células HeLa utilizando un dominio ZiF con dos dedos. Este protocolo es extensible a casi cualquier proteína capaz de la expresión soluble en Escherichia coli y casi cualquier tipo de célula de mamífero. Resultados esperados se proporcionan y también se discuten estrategias para maximizar el rendimiento de este sistema.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>XmaI</td>
			<td>New England Biolabs</td>
			<td>R0180L</td>
			<td></td>
		</tr>
		<tr>
			<td>SacI</td>
			<td>New England Biolabs</td>
			<td>R0156L</td>
			<td></td>
		</tr>
		<tr>
			<td>Expand High Fidelity PCR system</td>
			<td>Roche</td>
			<td>11759078001</td>
			<td></td>
		</tr>
		<tr>
			<td>dNTPs</td>
			<td>New England Biolabs</td>
			<td>N0446S</td>
			<td></td>
		</tr>
		<tr>
			<td>4-20% Tris-Glycine Mini protein gels, 1.5 mm, 10 wells</td>
			<td>Life Technologies</td>
			<td>EC6028BOX</td>
			<td></td>
		</tr>
		<tr>
			<td>2x Laemmli Sample Buffer</td>
			<td>BioRad</td>
			<td>161-0737</td>
			<td></td>
		</tr>
		<tr>
			<td>T4 DNA Ligase</td>
			<td>Life Technologies</td>
			<td>15224-017</td>
			<td></td>
		</tr>
		<tr>
			<td>BL21 (DE3) Competent E. coli</td>
			<td>New England Biolabs</td>
			<td>C2527I</td>
			<td></td>
		</tr>
		<tr>
			<td>IPTG</td>
			<td>Thermo Scientific</td>
			<td>R0391</td>
			<td></td>
		</tr>
		<tr>
			<td>Zinc Chloride</td>
			<td>Sigma-Aldrich</td>
			<td>208086-5G</td>
			<td></td>
		</tr>
		<tr>
			<td>Kanamycin Sulfate</td>
			<td>Fisher Scientific</td>
			<td>BP906-5</td>
			<td></td>
		</tr>
		<tr>
			<td>Glucose</td>
			<td>Sigma-Aldrich</td>
			<td>G8270-100G</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris Base</td>
			<td>Fisher Scientific</td>
			<td>BP152-25</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Chloride</td>
			<td>Sigma-Aldrich</td>
			<td>S9888-25G</td>
			<td></td>
		</tr>
		<tr>
			<td>DTT</td>
			<td>Fisher Scientific</td>
			<td>PR-V3151&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>PMSF</td>
			<td>Thermo Scientific</td>
			<td>36978</td>
			<td></td>
		</tr>
		<tr>
			<td>Ni-NTA Agarose Resin</td>
			<td>QIAGEN</td>
			<td>30210</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycerol</td>
			<td>Sigma-Aldrich</td>
			<td>G5516-500ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Imidazole</td>
			<td>Sigma-Aldrich</td>
			<td>I5513-25G</td>
			<td></td>
		</tr>
		<tr>
			<td>Amicon Ultra-15 Centrifugal Filter Units</td>
			<td>EMO Millipore</td>
			<td>UFC900324</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Life Technologies</td>
			<td>11966-025</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Life Technologies</td>
			<td>10437-028</td>
			<td></td>
		</tr>
		<tr>
			<td>Antibiotic-Antimycotic&#160;</td>
			<td>Life Technologies</td>
			<td>15240-062</td>
			<td></td>
		</tr>
		<tr>
			<td>24-Well Flat Bottom Plate</td>
			<td>Sigma-Aldrich</td>
			<td>CLS3527-100EA</td>
			<td></td>
		</tr>
		<tr>
			<td>Poly-Lysine</td>
			<td>Sigma-Aldrich</td>
			<td>P7280</td>
			<td></td>
		</tr>
		<tr>
			<td>DPBS, No Calcium, No Magnesium</td>
			<td>Life Technologies</td>
			<td>21600010</td>
			<td></td>
		</tr>
		<tr>
			<td>Heparan Sulfate</td>
			<td>Sigma-Aldrich</td>
			<td>H4777</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin</td>
			<td>Life Technologies</td>
			<td>25300054</td>
			<td></td>
		</tr>
		<tr>
			<td>Hela cells</td>
			<td>ATCC</td>
			<td>CCL-2</td>
			<td></td>
		</tr>
		<tr>
			<td>Nano Drop ND-1000 spectrophotometer&#160;</td>
			<td>Thermo Fisher Scientific</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>QIAquick PCR Purification Kit</td>
			<td>QIAGEN</td>
			<td>28104</td>
			<td></td>
		</tr>
		<tr>
			<td>QIAquick Gel Extraction Kit</td>
			<td>QIAGEN</td>
			<td>28704</td>
			<td></td>
		</tr>
	</tbody>
</table>

direct protein delivery to mammalian cells using cell-permeable cys2-his2 zinc-finger domains

Due to their modularity and ability to be reprogrammed to recognize a wide range of DNA sequences, Cys2-His2 zinc-finger DNA-binding domains have emerged as useful tools for targeted genome engineering. Like many other DNA-binding proteins, zinc-fingers also possess the innate ability to cross cell membranes. We recently demonstrated that this intrinsic cell-permeability could be leveraged for intracellular protein delivery. Genetic fusion of zinc-finger motifs leads to efficient transport of protein and enzyme cargo into a broad range of mammalian cell types. Unlike other protein transduction technologies, delivery via zinc-finger domains does not inhibit enzyme activity and leads to high levels of cytosolic delivery. Here a detailed step-by-step protocol is presented for the implementation of zinc-finger technology for protein delivery into mammalian cells. Key steps for achieving high levels of intracellular zinc-finger-mediated delivery are highlighted and strategies for maximizing the performance of this system are discussed.

Con dos dedos proteínas de fusión ZiF-EmGFP pueden expresarse en E. coli con&gt; 95% de homogeneidad y altos rendimientos (&gt; 25 mg / ml) (Figura 2). En general, de uno y dos dedos proteínas de fusión ZIF pueden ser producidos en cantidades casi idénticas a las de la proteína no modificada de tipo salvaje. Sin embargo, en algunos contextos, proteínas de fusión ZiF de cinco y seis dedos no son capaces de ser producidos en rendimientos suficientemente altos para aplicaciones posteriores...

Watch this Scientific Journal Video about Direct Protein Delivery to Mammalian Cells Using Cell-permeable Cys2-His2 Zinc-finger Domains at JoVE.com

Direct Protein Delivery to Mammalian Cells Using Cell-permeable Cys2-His2 Zinc-finger Domains

Dominios de dedos de zinc son células permeable intrínseca y capaz de mediar la entrega de proteínas en una amplia gama de tipos de células de mamíferos. Aquí, se presenta un protocolo detallado paso a paso para la implementación de la tecnología zinc-finger para la entrega de proteínas intracelulares.

Proteína entrega directa de células de mamíferos Utilización de Células permeable Cys<sub&gt; 2</sub&gt; -Su<sub&gt; 2</sub&gt; Dominios de zinc-finger

Due to their modularity and ability to be reprogrammed to recognize a wide range of DNA sequences, Cys2-His2 zinc-finger DNA-binding domains have emerged ...

direct-protein-delivery-to-mammalian-cells-using-cell-permeable-cys2

Research

JoVE Journal

Biology

Direct Protein Delivery to Mammalian Cells Using Cell-permeable Cys2-His2 Zinc-finger Domains

9.8K vistas.  The Scripps Research Institute.  Dominios de dedos de zinc son células permeable intrínseca y capaz de mediar la entrega de proteínas en una amplia gama de tipos de células de mamíferos. Aquí, se presenta un protocolo detallado paso a paso para la implementación de la tecnología zinc-finger para la entrega de proteínas intracelulares. 

Video: Direct Protein Delivery to Mammalian Cells Using Cell-permeable Cys2-His2 Zinc-finger Domains

Long-term Imaging Mammalian Cells using Wide-Field Microscopy

A largo plazo de imágenes usando las células de mamíferos Wide-Field Microscopía

Investigación

Biología

Trypsinizing and Subculturing Mammalian Cells

As cells reach confluency, they must be subcultured or passaged. Failure to subculture confluent cells results in reduced mitotic index and eventually in cell death. The first step in subculturing is to detach cells from the surface of the primary culture vessel by trypsinization or mechanical means. The resultant cell suspension is then subdivided, or reseeded, into fresh cultures. Secondary cultures are checked for growth and fed periodically, and may be subsequently subcultured to produce tertiary cultures. The time between passaging of cells varies with the cell line and depends on the growth rate.

As cells reach confluency, they must be subcultured or passaged. This video will demonstrate a procedure for subculturing both adherent and suspension cells.

Trypsinizing Siembra y células de mamífero

As cells reach confluency, they must be subcultured or passaged. Failure to subculture confluent cells results in reduced mitotic index and eventually in ...

Engineering Cell-permeable Protein

The protein transduction technique enables the direct delivery of biologically active material into mammalian cells [for review see 1,2]. For this one can make use of the translocating ability of so-called cell penetrating peptides (CPPs), also designated as protein transduction domains (PTDs). The TAT-CPP derived from the human immunodeficiency virus type 1 (HIV-1) Tat (trans-activator of transcription) protein has been widely used. The positively charged TAT promotes cell permeability thereby overcoming the barriers of the cellular membrane by endocytosis or/and direct membrane penetration2. In combination with a nuclear localization signal (NLS) fusion proteins are able to enter the nucleus exhibiting functionality. Our video presentation demonstrates, as an exemplification for the engineering of cell-permeable proteins, the construction, production and application of a cell-permeable version of the DNA-modifying enzyme Cre.
Cre is a site-specific recombinase that is able to recognize and recombine 34 base pair loxP sites in mammalian cells in vitro and in vivo. Therefore the Cre/loxP system is widely used to conditionally induce mutations in the genome of living cells3,4. The delivery of active Cre recombinase to cells, however, represents a limitation.
We describe the pSESAME vector system, which allows a direct insertion of the gene-of-interest and provides a platform to rapidly clone different domains and tags used within the vector in a convenient and standardized manner. Rearranging of the different tags has been shown to modify the biochemical properties of the fusion proteins providing a possibility to achieve higher yield and better solubility. We demonstrate how to express and purify recombinant cell-permeant proteins in and from E. coli. The functionality of the recombinant Cre protein is finally validated in cell culture by assessing its intracellular recombinase activity.

Protein transduction enables the direct delivery of biologically active proteins into cells. In contrast to conventional methods such as DNA transfection or viral transduction this non-invasive paradigm allows highly efficient cellular manipulation in a titratable manner circumventing cellular toxicity and the risk of oncogenic transformation by permanent genetic modification.

Ingeniería penetran en las células de proteínas

The protein transduction technique enables the direct delivery of biologically active material into mammalian cells [for review see 1,2]. For this one can ...

Using the GELFREE 8100 Fractionation System for Molecular Weight-Based Fractionation with Liquid Phase Recovery

The GELFREE 8100 Fractionation System is a novel protein fractionation system designed to maximize protein recovery during molecular weight based fractionation. The system is comprised of single-use, 8-sample capacity cartridges and a benchtop GELFREE Fractionation Instrument. During separation, a constant voltage is applied between the anode and cathode reservoirs, and each protein mixture is electrophoretically driven from a loading chamber into a specially designed gel column gel. Proteins are concentrated into a tight band in a stacking gel, and separated based on their respective electrophoretic mobilities in a resolving gel. As proteins elute from the column, they are trapped and concentrated in liquid phase in the collection chamber, free of the gel. The instrument is then paused at specific time intervals, and fractions are collected using a pipette. This process is repeated until all desired fractions have been collected. If fewer than 8 samples are run on a cartridge, any unused chambers can be used in subsequent separations.
This novel technology facilitates the quick and simple separation of up to 8 complex protein mixtures simultaneously, and offers several advantages when compared to previously available fractionation methods. This system is capable of fractionating up to 1mg of total protein per channel, for a total of 8mg per cartridge. Intact proteins over a broad mass range are separated on the basis of molecular weight, retaining important physiochemical properties of the analyte. The liquid phase entrapment provides for high recovery while eliminating the need for band or spot cutting, making the fractionation process highly reproducible1.

The accompanying video describes the use of the GELFREE 8100 Fractionation System, which partitions complex protein samples on the basis of molecular weight and recovers the fractions in liquid phase. The video describes how the technology works, how it is used, and provides resultant data, with polyacrylamide gel electrophoresis analysis of fractionated bovine liver homogenate.

Utilizando el sistema de fraccionamiento GELFREE 8100 de peso molecular basado en Fraccionamiento con la recuperación de la fase líquida

The GELFREE 8100 Fractionation System is a novel protein fractionation system designed to maximize protein recovery during molecular weight based ...

Genome Editing with CompoZr Custom Zinc Finger Nucleases (ZFNs)

Genome editing is a powerful technique that can be used to elucidate gene function and the genetic basis of disease. Traditional gene editing methods such as chemical-based mutagenesis or random integration of DNA sequences confer indiscriminate genetic changes in an overall inefficient manner and require incorporation of undesirable synthetic sequences or use of aberrant culture conditions, potentially confusing biological study. By contrast, transient ZFN expression in a cell can facilitate precise, heritable gene editing in a highly efficient manner without the need for administration of chemicals or integration of synthetic transgenes.
Zinc finger nucleases (ZFNs) are enzymes which bind and cut distinct sequences of double-stranded DNA (dsDNA). A functional CompoZr ZFN unit consists of two individual monomeric proteins that bind a DNA "half-site" of approximately 15-18 nucleotides (see Figure 1). When two ZFN monomers "home" to their adjacent target sites the DNA-cleavage domains dimerize and create a double-strand break (DSB) in the DNA.1 Introduction of ZFN-mediated DSBs in the genome lays a foundation for highly efficient genome editing. Imperfect repair of DSBs in a cell via the non-homologous end-joining (NHEJ) DNA repair pathway can result in small insertions and deletions (indels). Creation of indels within the gene coding sequence of a cell can result in frameshift and subsequent functional knockout of a gene locus at high efficiency.2 While this protocol describes the use of ZFNs to create a gene knockout, integration of transgenes may also be conducted via homology-directed repair at the ZFN cut site.
The CompoZr Custom ZFN Service represents a systematic, comprehensive, and well-characterized approach to targeted gene editing for the scientific community with ZFN technology. Sigma scientists work closely with investigators to 1) perform due diligence analysis including analysis of relevant gene structure, biology, and model system pursuant to the project goals, 2) apply this knowledge to develop a sound targeting strategy, 3) then design, build, and functionally validate ZFNs for activity in a relevant cell line. The investigator receives positive control genomic DNA and primers, and ready-to-use ZFN reagents supplied in both plasmid DNA and in-vitro transcribed mRNA format. These reagents may then be delivered for transient expression in the investigator&#x2019;s cell line or cell type of choice. Samples are then tested for gene editing at the locus of interest by standard molecular biology techniques including PCR amplification, enzymatic digest, and electrophoresis. After positive signal for gene editing is detected in the initial population, cells are single-cell cloned and genotyped for identification of mutant clones/alleles.

Genome Editing with CompoZr Custom Zinc Finger Nucleases ZFNs

The CompoZr Custom Zinc-Finger Nuclease (ZFN) Service enables precise genome editing in any organism or cell line at any locus defined by the user. This article describes the process for the design, manufacture, validation and implementation of the CompoZr Custom ZFN Service.

Genoma Edición con CompoZr nucleasas de dedos de zinc personalizados (ZFNs)

Genome editing is a powerful technique that can be used to elucidate gene function and the genetic basis of disease. Traditional gene editing methods such ...

siRNA Screening to Identify Ubiquitin and Ubiquitin-like System Regulators of Biological Pathways in Cultured Mammalian Cells

Post-translational modification of proteins with ubiquitin and ubiquitin-like molecules (UBLs) is emerging as a dynamic cellular signaling network that regulates diverse biological pathways including the hypoxia response, proteostasis, the DNA damage response and transcription.&#160; To better understand how UBLs regulate pathways relevant to human disease, we have compiled a human siRNA &#8220;ubiquitome&#8221; library consisting of 1,186 siRNA duplex pools targeting all known and predicted components of UBL system pathways. This library can be screened against a range of cell lines expressing reporters of diverse biological pathways to determine which UBL components act as positive or negative regulators of the pathway in question.&#160; Here, we describe a protocol utilizing this library to identify ubiquitome-regulators of the HIF1A-mediated cellular response to hypoxia using a transcription-based luciferase reporter.&#160; An initial assay development stage is performed to establish suitable screening parameters of the cell line before performing the screen in three stages: primary, secondary and tertiary/deconvolution screening. &#160;The use of targeted over whole genome siRNA libraries is becoming increasingly popular as it offers the advantage of reporting only on members of the pathway with which the investigators are most interested.&#160; Despite inherent limitations of siRNA screening, in particular false-positives caused by siRNA off-target effects, the identification of genuine novel regulators of the pathways in question outweigh these shortcomings, which can be overcome by performing a series of carefully undertaken control experiments.

Here, we describe a methodology to perform a targeted siRNA &#8220;ubiquitome&#8221; screen to identify novel ubiquitin and ubiquitin-like regulators of the HIF1A-mediated cellular response to hypoxia.&#160; This can be adapted to any biological pathway where a robust read out of reporter activity is available.

Screening siRNA para identificar ubiquitina y reguladores del sistema ubiquitina-como de las vías biológicas en células de mamífero en cultivo

Post-translational modification of proteins with ubiquitin and ubiquitin-like molecules (UBLs) is emerging as a dynamic cellular signaling network that ...

Identification of Protein Interaction Partners in Mammalian Cells Using SILAC-immunoprecipitation Quantitative Proteomics

Quantitative proteomics combined with immuno-affinity purification, SILAC immunoprecipitation, represent a powerful means for the discovery of novel protein:protein interactions. By allowing the accurate relative quantification of protein abundance in both control and test samples, true interactions may be easily distinguished from experimental contaminants. Low affinity interactions can be preserved through the use of less-stringent buffer conditions and remain readily identifiable. This protocol discusses the labeling of tissue culture cells with stable isotope labeled amino acids, transfection and immunoprecipitation of an affinity tagged protein of interest, followed by the preparation for submission to a mass spectrometry facility. This protocol then discusses how to analyze and interpret the data returned from the mass spectrometer in order to identify cellular partners interacting with a protein of interest. As an example this technique is applied to identify proteins binding to the eukaryotic translation initiation factors: eIF4AI and eIF4AII.

SILAC immunoprecipitation experiments represent a powerful means for discovering novel protein:protein interactions. By allowing the accurate relative quantification of protein abundance in both control and test samples, true interactions may be easily distinguished from experimental contaminants, and low affinity interactions preserved through use of less-stringent buffer conditions.

Identificación de proteínas compañeros de interacción en células de mamífero utilizando SILAC-inmunoprecipitación cuantitativos Proteómica

Quantitative proteomics combined with immuno-affinity purification, SILAC immunoprecipitation, represent a powerful means for the discovery of novel ...

Zinc-finger Nuclease Enhanced Gene Targeting in Human Embryonic Stem Cells

One major limitation with current human embryonic stem cell (ESC) differentiation protocols is the generation of heterogeneous cell populations.&#160;These cultures contain the cells of interest, but are also contaminated with undifferentiated ESCs, non-neural derivatives and other neuronal subtypes.&#160; This limits their use in in vitro and in vivo applications, such as in vitro modeling for drug discovery or cell replacement therapy. To help overcome this, reporter cell lines, which offer a means to visualize, track and isolate cells of interest, can be engineered.&#160;However, to achieve this in human embryonic stem cells via conventional homologous recombination is extremely inefficient.&#160;This protocol describes targeting of the Pituitary homeobox 3 (PITX3) locus in human embryonic stem cells using custom designed zinc-finger nucleases, which introduce site-specific double-strand DNA breaks, together with a PITX3-EGFP-specific DNA donor vector. Following the generation of the PITX3 reporter cell line, it can then be differentiated using published protocols for use in studies such as in vitro Parkinson&#8217;s disease modeling or cell replacement therapy.

Reporter cell lines offer a means to visualize, track and isolate cells of interest from heterogeneous populations.&#160;However, gene-targeting using conventional homologous recombination in human embryonic stem cells is extremely inefficient. Herein, we describe targeting CNS midbrain specific transcription factor PITX3 locus with EGFP using zinc-finger nuclease enhanced homologous recombination.

Nucleasa dedo de zinc-gen mejorada en células madre embrionarias humanas

One major limitation with current human embryonic stem cell (ESC) differentiation protocols is the generation of heterogeneous cell ...

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.

El examen de las proteínas unidas al ADN naciente en células de mamífero utilizando BrdU-chip-Slot-occidental Técnica

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 ...

A Graphical User Interface for Software-assisted Tracking of Protein Concentration in Dynamic Cellular Protrusions

Filopodia are dynamic, finger-like cellular protrusions associated with migration and cell-cell communication. In order to better understand the complex signaling mechanisms underlying filopodial initiation, elongation and subsequent stabilization or retraction, it is crucial to determine the spatio-temporal protein activity in these dynamic structures. To analyze protein function in filopodia, we recently developed a semi-automated tracking algorithm that adapts to filopodial shape-changes, thus allowing parallel analysis of protrusion dynamics and relative protein concentration along the whole filopodial length. Here, we present a detailed step-by-step protocol for optimized cell handling, image acquisition and software analysis. We further provide instructions for the use of optional features during image analysis and data representation, as well as troubleshooting guidelines for all critical steps along the way. Finally, we also include a comparison of the described image analysis software with other programs available for filopodia quantification. Together, the presented protocol provides a framework for accurate analysis of protein dynamics in filopodial protrusions using image analysis software.

We present a software solution for semi-automated tracking of relative protein concentration along the length of dynamic cellular protrusions.

Una interfaz gráfica de usuario para el seguimiento asistido por software de la concentración de proteínas en protones dinámicos celulares

Filopodia are dynamic, finger-like cellular protrusions associated with migration and cell-cell communication. In order to better understand the complex ...