Estamos agradecidos con el Centro de Cáncer de la Universidad Estatal de Ohio Integral para proporcionar fondos para este trabajo.

1. Línea celular con un sustrato de recombinación integrado <ol><li> Las células HeLa fueron transformados de forma estable con el PDR-GFP (plásmido 7 regalo de M. Jasin, Memorial Sloan-Kettering Cancer Center) y se mantiene en puromicina para la selección de los transformantes. </li><li> Las células se pasan en DMEM / FBS, que contiene 10% de suero fetal bovino, 2 mM Glutamax, 1 mM piruvato sódico, a la penicilina / estreptomicina (100 U / mL/0.1 mg / ml), y 1,5 puromicina ug / mL. </li></ol> El sustrato de la recombinación en el PDR-GFP plásmido, y en los transformantes estables, contiene dos alelos inactivos de las buenas prácticas agrarias (Figura 1). Un alelo está inactivo debido a la inclusión en su secuencia de codificación de la página 18 pb restricción de reconocimiento de enzimas para la I-SCE. Expresión de I-SCE en estas células crea un salto de ADN de doble cadena (DSB). Hay un segundo alelo de GFP inactivo en este ADN, pero la parte de su secuencia relacionada con el sitio I-SCE en el primer alelo es de tipo salvaje. Este alelo GFP segundo puede funcionar como una secuencia de donantes para la reparación de recombinación homóloga del OSD y se traduciría en un alelo GFP activo, que es fácilmente detectado por citometría de flujo. Por otra parte, el OSD podría ser reparado por la unión de extremos no homólogos, lo que destruiría el sitio de restricción I-SCEI y daría lugar a las buenas prácticas agrarias-negativos células. El nivel de recombinación homóloga en una celda se determina contando el porcentaje de las buenas prácticas agrarias de células positivas. Esta línea celular, pHeLa-DR13-9, fue seleccionado entre todos los clones de la señal cero GFP de fondo y un porcentaje relativamente alto de las buenas prácticas agrarias de células positivas a la transfección con el I-SCE expresar pCBASce plásmido. (El pCBASce y su control de vectores, pCAGGS, fueron suministrados por M. Jasin del Centro del Cáncer Memorial Sloan-Kettering.) La línea HeLa-DR13-9 está disponible de células de los autores que lo soliciten. 2. Transfección de BRCA1 agotan endógenos y añadir de nuevo mutante BRCA1. <ol><li> El día antes de la transfección primero (por lo general un martes): Comience con un plato de 10 cm de HeLa-DR13-9 células de al menos el 90% confluente. </li><li> Trypsinize en 1 ml y detener la reacción mediante la adición de 9 ml DMEM / FBS, se mezcla bien con la pipeta. </li><li> Añadir 40 l de células con tripsina por pocillo de una placa de 24 pocillos de 0,5 ml de DME / FBS. </li><li> Día 1 transfecciones (miércoles): las células deben ser confluentes ~ 40-50%, aunque mucho menos empezar de nuevo. Transfectar mediante la mezcla de: 5 siRNA pmol + 0,3 ug-mutante BRCA1 expresión de ADN plásmido en el 12,5 l Opti-MEM, y 0,5 l Lipofectamine 2000 en 12,5 l Opti-MEM. Proceder a la transfección de acuerdo con el protocolo Lipofectamine (Invitrogen). </li><li> Vuelva a colocar los medios de comunicación 4-6 horas más tarde. El siRNA se utiliza para agotar BRCA1 se basa en la secuencia que especifica GCUCCUCUCACUCUUCAGU BRCA1 3&#39;-UTR secuencias de 80.780 a 80.798 (número de acceso AY273801). </li><li> Día 2: Traslado de las células por trypsinizing de la placa y 24 a una placa de 6 pocillos. </li><li> Día 3 transfecciones: 25 pmol siRNA + 0,75 ug-mutante BRCA1 expresión de ADN plásmido + 0,75 ug pCBASce (o pCAGGS control de vectores) en el 62,5 l Opti-MEM y 2,5 l Lipofectamine 2000 en el 62,5 l Opti-MEM. Vuelva a colocar los medios de comunicación 4-6 horas más tarde. </li></ol> 3. Análisis de los transfectantes. <ol><li> El día 6, trypsinize células de cada pocillo y dejar en 1 ml de DMEM / FBS. Análisis de las células para la expresión de GFP se puede hacer en los días 5 y 7, pero nos encontramos con que el día 6 que funciona mejor. </li><li> Analizar 10.000 células por pocillo por citometría de flujo. Utilizamos un Becton Dickinson FACSCalibur instrumento en la Ohio State University Comprehensive Cancer Center de Citometría de recursos compartidos de análisis. Las células individuales, en vivo son cerradas con los parámetros de dispersión frontal y lateral. Las buenas prácticas agrarias de células positivas se detectan mediante el detector de fluorescencia de GFP (FL1), y los resultados se presentan en forma de histograma (Figura 2, abajo). </li><li> Izquierda sobre las células puede ser replated para la fotografía de microscopía de fluorescencia, si lo desea. </li></ol> 4. Los resultados representativos: BRCA1 regula la vía para la reparación de roturas de doble cadena de ADN por recombinación homóloga 6,11. Las células que han sido sometidos a reparación por recombinación homóloga son fácilmente detectados por microscopía de fluorescencia (Figura 2, arriba). El agotamiento de los resultados de BRCA1 en una disminución de la GFP-positivas detectadas células (Figura 2, derecha). La salida de la FACSCalibur es un histograma con intensidad por las buenas prácticas agrarias de células en el eje X y el número de células en el eje Y (Figura 2, abajo). En un conjunto típico de los resultados de un experimento único, el 15,5% de las células fueron GFP-positivo después de la I-II CPE expresión y el control de agotamiento de RNAi (Figura 3). En comparación, el agotamiento de los genes BRCA1 y añadir de nuevo vector GFP reduce la conversión de hasta el 0,7%. Add-back de tipo salvaje BRCA1 o de los genes BRCA1 (I21V) totalmente restaurada la recombinación homóloga, según lo detectado por la obtención de un 15-16% de las células GFP positivos. Add-back de los genes BRCA1 (M18T) mutante (Figura 3, carril 5) tuvo un efecto similar a la extensión posterior de la lucha contra los vectores. Desde esta variante expresa en niveles similares a la proteína endógena 9, los intérpretes de los genes BRCA1 (M18T) la variante no es funcional en la recombinación homóloga. Por lo general tienen un 10-20% de las células convierten a GFP-positivo después de la transfección de la expresión I-SCE plásmido. El agotamiento de los genes BRCA1 y reproducen reduce la conversión de las buenas prácticas agrarias de células positivas en alrededor de 8.10 veces. Aunque los porcentajes reales de las buenas prácticas agrarias de células positivas pueden cambiar de un experimento a experimento, la proporción de las buenas prácticas agrarias de células positivas tras el agotamiento de BRCA1 en relación con el agotamiento de control dentro de un experimento es bastante consistente. Normalizamos los resultados mediante el establecimiento de la tasa de conversión sin restricciones a 100% y los resultados de expresar con el agotamiento BRCA1 y añadir la espalda-en forma de porcentajes. De esta forma, podemos combinar múltiples experimentos, y la variación experimental no es muy alta. Añadir de nuevo de los mutantes BRCA1 han arrojado resultados sólidos: cada variante BRCA1 o ha restaurado completamente homóloga actividad de recombinación o ha tenido ningún efecto. Una consideración importante en la obtención de estos resultados es que nuestras condiciones de transfección producir niveles de la proteína BRCA1 que aproximadamente igual a la concentración de la proteína endógena en las células no tratadas. Demasiado alto de una sobreexpresión de un mutante incluso defectuosa del gen BRCA1 pueden dar lugar a algún tipo de actividad de recombinación homóloga positivo. Un problema que tuvimos que superar fue la toxicidad de la transfección. El agotamiento de los genes BRCA1 se puede retrasar el crecimiento celular y el equilibrio de ADN Lipofectamine-2000 y el plásmido en relación con el número de células en el plato debe mantenerse en un nivel constante. ADN demasiado Lipofectamine plásmido o reducirá el número de células, y el número de células no serán suficientes para obtener resultados fiables de la citometría de flujo. El saldo de los reactivos Lipofectamine plásmido y contra el número de células transfectadas es muy importante para mantener la viabilidad de las células. <img src="/files/ftp_upload/2468/2468fig1.jpg" alt="Figura 1" /> Figura 1. El sustrato recombinación. La estrategia que ha sido desarrollado por el grupo de M. Jasin 7,12 se representa. El PDR-GFP plásmido contiene dos alelos defectuosos de las buenas prácticas agrarias, una de las cuales contiene un sitio de restricción de endonucleasa I-SCE. Una línea de células HeLa derivada contiene este sustrato de ADN integrados en un solo sitio en el genoma de 9, y la reparación de activos de la ruptura del ADN de doble cadena en el sitio I-II CPE por los resultados de recombinación homóloga en la conversión de un alelo de GFP-positivas. <img src="/files/ftp_upload/2468/2468fig2.jpg" alt="Figura 2" /> Figura 2. Detección de las buenas prácticas agrarias de células positivas por microscopía de fluorescencia. Las células fueron comprobados de acuerdo con este protocolo, y el control-agotadas las células se activa en la recombinación homóloga, detectada por un alto porcentaje de células con expresión de las buenas prácticas agrarias (izquierda). El agotamiento de los genes BRCA1 en los resultados de RNAi en una fuerte disminución en el número de células positivas para GFP (derecha). <img src="/files/ftp_upload/2468/2468fig3.jpg" alt="Figura 3" /> Figura 3. Los resultados de un add-back de los mutantes missense BRCA1. Los resultados de un solo experimento se muestran en el histograma. En ausencia de transfectadas I-SCE expresar plásmido (carril 1) no hay células positivas GFP-detectados. Los resultados en los carriles 2-6 se tomaron todas las células en la que el I-SCE expresar plásmido se transfectadas en el día 3. En las células que se agotaron por un gen irrelevante (luciferasa) y con el vector add-back, el 15,5% de las células fueron GFP-positivo (carril 2). El agotamiento de los genes BRCA1 y vector add-back revela el efecto de la pérdida de BRCA1 en la recombinación homóloga (calle 3). Los efectos del agotamiento de los genes BRCA1 y add-back de tipo salvaje BRCA1 (calle 4), BRCA1 (M18T) (carril 5), y BRCA1 (I21V) (carril 6) se muestran. Estos resultados son de un solo experimento, y se puede combinar con al menos dos experimentos repetidos más con el fin de obtener una medida de la recombinación homóloga con el apoyo de una proteína mutante BRCA1 dado.

<ol>
	<li>Carvalho, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determination+of+cancer+risk+associated+with+germ+line+BRCA1+missense+variants+by+functional+analysis.">Determination of cancer risk associated with germ line BRCA1 missense variants by functional analysis.</a> Cancer Res. 67, 1494-1501 (2007).</li><li>Couch, F. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+epidemiology+of+BRCA1.">Genetic epidemiology of BRCA1.</a> Cancer Biol Ther. 3, 509-514 (2004).</li><li>Couch, F. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessment+of+functional+effects+of+unclassified+genetic+variants.">Assessment of functional effects of unclassified genetic variants.</a> Hum Mutat. 29, 1314-1326 (2008).</li><li>Tomlinson, G. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+a+breast+cancer+cell+line+derived+from+a+germ-line+BRCA1+mutation+carrier.">Characterization of a breast cancer cell line derived from a germ-line BRCA1 mutation carrier.</a> Cancer Res. 58, 3237-3242 (1998).</li><li>Ruffner, H., Joazeiro, C. A., Hemmati, D., Hunter, T., Verma, I. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cancer-predisposing+mutations+within+the+RING+domain+of+BRCA1:+loss+of+ubiquitin+protein+ligase+activity+and+protection+from+radiation+hypersensitivity.">Cancer-predisposing mutations within the RING domain of BRCA1: loss of ubiquitin protein ligase activity and protection from radiation hypersensitivity.</a> Proc Natl Acad Sci U S A. 98, 5134-5139 (2001).</li><li>Moynahan, M. E., Chiu, J. W., Koller, B. H., 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=Brca1+controls+homology-directed+DNA+repair.">Brca1 controls homology-directed DNA repair.</a> Mol Cell. 4, 511-518 (1999).</li><li>Nakanishi, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+Fanconi+anemia+monoubiquitination+pathway+promotes+homologous+DNA+repair.">Human Fanconi anemia monoubiquitination pathway promotes homologous DNA repair.</a> Proc Natl Acad Sci U S A. 102, 1110-1115 (2005).</li><li>Pierce, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Double-strand+breaks+and+tumorigenesis.">Double-strand breaks and tumorigenesis.</a> Trends Cell Biol. 11, 52-59 (2001).</li><li>Ransburgh, D. J., Chiba, N., Ishioka, C., Toland, A. E., Parvin, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+breast+tumor+mutations+in+BRCA1+that+abolish+its+function+in+homologous+DNA+recombination.">Identification of breast tumor mutations in BRCA1 that abolish its function in homologous DNA recombination.</a> Cancer Res. 70, 988-995 (2010).</li><li>Easton, D. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+systematic+genetic+assessment+of+1,433+sequence+variants+of+unknown+clinical+significance+in+the+BRCA1+and+BRCA2+breast+cancer-predisposition+genes.">A systematic genetic assessment of 1,433 sequence variants of unknown clinical significance in the BRCA1 and BRCA2 breast cancer-predisposition genes.</a> Am J Hum Genet. 81, 873-883 (2007).</li><li>Snouwaert, J. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=BRCA1+deficient+embryonic+stem+cells+display+a+decreased+homologous+recombination+frequency+and+an+increased+frequency+of+non-homologous+recombination+that+is+corrected+by+expression+of+a+brca1+transgene.">BRCA1 deficient embryonic stem cells display a decreased homologous recombination frequency and an increased frequency of non-homologous recombination that is corrected by expression of a brca1 transgene.</a> Oncogene. 18, 7900-7907 (1999).</li><li>Pierce, A. J., Hu, P., Han, M., Ellis, N., 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=Ku+DNA+end-binding+protein+modulates+homologous+repair+of+double-strand+breaks+in+mammalian+cells.">Ku DNA end-binding protein modulates homologous repair of double-strand breaks in mammalian cells.</a> Genes Dev. 15, 3237-3242 (2001).</li><li>Spearman, A. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clinically+applicable+models+to+characterize+BRCA1+and+BRCA2+variants+of+uncertain+significance.">Clinically applicable models to characterize BRCA1 and BRCA2 variants of uncertain significance.</a> J Clin Oncol. 26, 5393-5400 (2008).</li><li>Sweet, K., Senter, L., Pilarski, R., Wei, L., Toland, A. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+BRCA1+ring+finger+variants+of+uncertain+significance.">Characterization of BRCA1 ring finger variants of uncertain significance.</a> Breast Cancer Res Treat. 119, 737-743 (2009).</li></ol>

La ventaja de este método es que se puede estudiar la función de la proteína BRCA1 en la regulación de la reparación del ADN por recombinación homóloga utilizando células que han de tipo salvaje BRCA1 endógena expresó. Hemos adoptado dos métodos para establecer de estudiar el proceso de recombinación homóloga y de la utilización de RNAi agotamiento para obtener el nuevo método para las pruebas de BRCA1 variantes de función en la reparación del ADN. La clave para el éxito de este método consistía en establecer una línea de células fácilmente transfectable, tales como HeLa, con el sustrato de la recombinación en el genoma de tal manera que se obtienen altos niveles de conversión de las buenas prácticas agrarias. Hemos tenido múltiples clones seleccionados de la transformación original con el fin de obtener una que no tenía ninguna señal de fondo GFP detectable, pero a la transfección de la expresión I-SCE plásmido tenía un nivel muy alto de conversión de las buenas prácticas agrarias. Con este método, se están investigando otros residuos específicos BRCA1 aminoácidos para la función en la recombinación homóloga. Junto con los conocimientos biológicos de este trabajo, estas conclusiones se pueden aplicar a la genética del cáncer clínico. Un alto porcentaje de mutaciones missense BRCA1 tienen un significado clínico desconocido ya que hay muchas variantes raras y no hay información suficiente para muchos de estos para determinar la asociación del cáncer mediante análisis de la segregación genética 3,10,13,14. Usando este método, las consecuencias de una variante específica de la función biológica de los genes BRCA1 en la regulación de la recombinación homóloga podría ser utilizado para informar a las mujeres que llevan una de estas variantes en su genoma.

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identifying the effects of brca1 mutations on homologous recombination using cells that express endogenous wild-type brca1

El análisis funcional de las mutaciones sin sentido puede ser complicado por la presencia en la célula de la proteína endógena. Estructura-función de los análisis de los genes BRCA1 se han complicado por la falta de una prueba sólida de la proteína de longitud completa BRCA1 y las dificultades inherentes a la utilización de líneas celulares que expresan la proteína BRCA1 hypomorphic 1,2,3,4,5. Hemos desarrollado un sistema mediante el cual se agotó la proteína BRCA1 endógeno en una célula de forma aguda por RNAi dirigidas a la 3&#39;-UTR del ARNm de BRCA1 y reemplazado por la co-transfección de un plásmido que expresa una variante BRCA1. Una de las ventajas de este procedimiento es que el silenciamiento aguda de la sustitución de los genes BRCA1 y simultánea permiten que las células crezcan sin mutaciones secundarias o adaptaciones que puedan surgir con el tiempo para compensar la pérdida de la función de BRCA1. Este agotamiento y el procedimiento add-back se llevó a cabo en una línea celular HeLa derivados que se ensayó con facilidad para la actividad de recombinación homóloga. El ensayo de recombinación homóloga se basa en un método previamente publicado por el cual se integra un sustrato de la recombinación en el genoma (Figura 1) 6,7,8,9. Este sustrato tiene la rara recombinación de corte I-II CPE sitio de la enzima de restricción dentro de un alelo inactivo GFP, y aguas abajo es un segundo alelo GFP inactivo. Transfección del plásmido que expresa la I-SCE ocasiona una ruptura de doble cadena, que pueden ser reparados por la recombinación homóloga, y si la recombinación homóloga no reparar la ruptura que crea un alelo GFP activo que es fácilmente marcado por citometría de flujo para la expresión de la proteína GFP . El agotamiento de los genes BRCA1 endógena produjo una reducción de 8 a 10 veces en la actividad de recombinación homóloga, y add-back de tipo salvaje función plásmido recombinación homóloga totalmente restaurada. Cuando los mutantes de puntos específicos de los genes BRCA1 longitud total se expresa a partir de co-transfectadas plásmidos, el efecto de los mutantes sin sentido específico se pudo calificar. A modo de ejemplo, la expresión de los genes BRCA1 (M18T) de proteína, una variante de significado desconocido clínica 10, se expresa en estas células, que no pudo restaurar BRCA1 dependientes de la recombinación homóloga. Por el contrario, la expresión de otra variante, también de significado desconocido, BRCA1 (I21V) totalmente restaurada BRCA1 dependiente de recombinación homóloga función. Esta estrategia de las pruebas de la función de los genes BRCA1 mutaciones sin sentido se ha aplicado a otro sistema biológico ensayo para la función del centrosoma (Kais y otros, observaciones no publicadas). En general, este enfoque es adecuado para el análisis de mutantes sin sentido en cualquier gen que se deben analizar recesiva.

Watch this Scientific Journal Video about Identifying the Effects of BRCA1 Mutations on Homologous Recombination using Cells that Express Endogenous Wild-type BRCA1 at JoVE.com

Identifying the Effects of BRCA1 Mutations on Homologous Recombination using Cells that Express Endogenous Wild-type BRCA1

Ofrecemos un método para probar variantes de BRCA1 en un ensayo de cultivo de tejidos para la reparación basada en la recombinación homóloga de ADN dañado por el agotamiento de la proteína BRCA1 endógena a partir de una célula utilizando ARN de interferencia y su sustitución por un mutante BRCA1 punto que contiene un cambio de codificación.

La identificación de los efectos de las mutaciones BRCA1 en recombinación homóloga utilizando células que expresan endógeno de tipo salvaje BRCA1

The functional analysis of missense mutations can be complicated by the presence in the cell of the endogenous protein. Structure-function analyses of the ...

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14.5K vistas.  The Ohio State University. Ofrecemos un método para probar variantes de BRCA1 en un ensayo de cultivo de tejidos para la reparación basada en la recombinación homóloga de ADN dañado por el agotamiento de la proteína BRCA1 endógena a partir de una célula utilizando ARN de interferencia y su sustitución por un mutante BRCA1 punto que contiene un cambio de codificación.

Video: Identifying the Effects of BRCA1 Mutations on Homologous Recombination using Cells that Express Endogenous Wild-type BRCA1

Studying the Effects of Matrix Stiffness on Cellular Function using Acrylamide-based Hydrogels

Tissue stiffness is an important determinant of cellular function, and changes in tissue stiffness are commonly associated with fibrosis, cancer and cardiovascular disease1-11. Traditional cell biological approaches to studying cellular function involve culturing cells on a rigid substratum (plastic dishes or glass coverslips) which cannot account for the effect of an elastic ECM or the variations in ECM stiffness between tissues. To model in vivo tissue compliance conditions in vitro, we and others use ECM-coated hydrogels. In our laboratory, the hydrogels are based on polyacrylamide which can mimic the range of tissue compliances seen biologically12. "Reactive" cover slips are generated by incubation with NaOH followed by addition of 3-APTMS. Glutaraldehyde is used to cross-link the 3-APTMS and the polyacrylamide gel. A solution of acrylamide (AC), bis-acrylamide (Bis-AC) and ammonium persulfate is used for the polymerization of the hydrogel. N-hydroxysuccinimide (NHS) is incorporated into the AC solution to crosslink ECM protein to the hydrogel. Following polymerization of the hydrogel, the gel surface is coated with an ECM protein of choice such as fibronectin, vitronectin, collagen, etc.
The stiffness of a hydrogel can be determined by rheology or atomic force microscopy (AFM) and adjusted by varying the percentage of AC and/or bis-AC in the solution12. In this manner, substratum stiffness can be matched to the stiffness of biological tissues which can also be quantified using rheology or AFM. Cells can then be seeded on these hydrogels and cultured based upon the experimental conditions required. Imaging of the cells and their recovery for molecular analysis is straightforward. For this article, we define soft substrata as those having elastic moduli (E) &lt;3000 Pascal and stiff substrata/tissues as those with E &gt;20,000 Pascal.

The effect of substrata stiffness on cellular function can be modeled in vitro using polyacrylamide hydrogels of varying compliances.

El estudio de los efectos de la matriz de rigidez en la función celular usando como base la acrilamida-hidrogeles

Tissue stiffness is an important determinant of cellular function, and changes in tissue stiffness are commonly associated with fibrosis, cancer and ...

Investigación

Biología

Analyzing the Function of Small GTPases by Microinjection of Plasmids into Polarized Epithelial Cells

Epithelial cells polarize their plasma membrane into biochemically and functionally distinct apical and basolateral domains where the apical domain faces the 'free' surfaces and the basolateral membrane is in contact with the substrate and neighboring cells. Both membrane domains are separated by tight junctions, which form a diffusion barrier. Apical-basolateral polarization can be recapitulated successfully in culture when epithelial cells such as Madin-Darby Canine Kidney (MDCK) cells are seeded at high density on polycarbonate filters and cultured for several days 1 2. Establishment and maintenance of cell polarity is regulated by an array of small GTPases of the Ras superfamily such as RalA, Cdc42, Rab8, Rab10 and Rab13 3 4 5 6 7. Like all GTPases these proteins cycle between an inactive GDP-bound state and an active GTP-bound state. Specific mutations in the nucleotide binding regions interfere with this cycling 8. For example, Rab13T22N is permanently locked in the GDP-form and thus dubbed 'dominant negative', whereas Rab13Q67L can no longer hydrolyze GTP and is thus locked in a 'dominant active' state 7. To analyze their function in cells both dominant negative and dominant active alleles of GTPases are typically expressed at high levels to interfere with the function of the endogenous proteins 9. An elegant way to achieve high levels of overexpression in a short amount of time is to introduce the plasmids encoding the relevant proteins directly into the nuclei of polarized cells grown on filter supports using microinjection technique. This is often combined with the co-injection of reporter plasmids that encode plasma membrane receptors that are specifically sorted to the apical or basolateral domain. A cargo frequently used to analyze cargo sorting to the basolateral domain is a temperature sensitive allele of the vesicular stomatitis virus glycoprotein (VSVGts045) 10. This protein cannot fold properly at 39&deg;C and will thus be retained in the endoplasmic reticulum (ER) while the regulatory protein of interest is assembled in the cytosol. A shift to 31&deg;C will then allow VSVGts045 to fold properly, leave the ER and travel to the plasma membrane 11. This chase is typically performed in the presence of cycloheximide to prevent further protein synthesis leading to cleaner results. Here we describe in detail the procedure of microinjecting plasmids into polarized cells and subsequent incubations including temperature shifts that allow a comprehensive analysis of regulatory proteins involved in basolateral sorting.

This article details the procedures involved in overexpression and analysis of small GTPases in polarized epithelial cells using microinjection technique.

El análisis de la función de las pequeñas GTPasas de la microinyección de plásmidos en células epiteliales polarizadas

Epithelial cells polarize their plasma membrane into biochemically and functionally distinct apical and basolateral domains where the apical domain faces ...

A PCR-based Genotyping Method to Distinguish Between Wild-type and Ornamental Varieties of Imperata cylindrica

Wild-type I. cylindrica (cogongrass) is one of the top ten worst invasive plants in the world, negatively impacting agricultural and natural resources in 73 different countries throughout Africa, Asia, Europe, New Zealand, Oceania and the Americas1-2. Cogongrass forms rapidly-spreading, monodominant stands that displace a large variety of native plant species and in turn threaten the native animals that depend on the displaced native plant species for forage and shelter. To add to the problem, an ornamental variety [I. cylindrica var. koenigii (Retzius)] is widely marketed under the names of Imperata cylindrica 'Rubra', Red Baron, and Japanese blood grass (JBG). This variety is putatively sterile and noninvasive and is considered a desirable ornamental for its red-colored leaves. However, under the correct conditions, JBG can produce viable seed (Carol Holko, 2009 personal communication) and can revert to a green invasive form that is often indistinguishable from cogongrass as it takes on the distinguishing characteristics of the wild-type invasive variety4 (Figure 1). This makes identification using morphology a difficult task even for well-trained plant taxonomists. Reversion of JBG to an aggressive green phenotype is also not a rare occurrence. Using sequence comparisons of coding and variable regions in both nuclear and chloroplast DNA, we have confirmed that JBG has reverted to the green invasive within the states of Maryland, South Carolina, and Missouri. JBG has been sold and planted in just about every state in the continental U.S. where there is not an active cogongrass infestation. The extent of the revert problem in not well understood because reverted plants are undocumented and often destroyed.
Application of this molecular protocol provides a method to identify JBG reverts and can help keep these varieties from co-occurring and possibly hybridizing. Cogongrass is an obligate outcrosser and, when crossed with a different genotype, can produce viable wind-dispersed seeds that spread cogongrass over wide distances5-7. JBG has a slightly different genotype than cogongrass and may be able to form viable hybrids with cogongrass. To add to the problem, JBG is more cold and shade tolerant than cogongrass8-10, and gene flow between these two varieties is likely to generate hybrids that are more aggressive, shade tolerant, and cold hardy than wild-type cogongrass. While wild-type cogongrass currently infests over 490 million hectares worldwide, in the Southeast U.S. it infests over 500,000 hectares and is capable of occupying most of the U.S. as it rapidly spreads northward due to its broad niche and geographic potential3,7,11. The potential of a genetic crossing is a serious concern for the USDA-APHIS Federal Noxious Week Program. Currently, the USDA-APHIS prohibits JBG in states where there are major cogongrass infestations (e.g., Florida, Alabama, Mississippi). However, preventing the two varieties from combining can prove more difficult as cogongrass and JBG expand their distributions. Furthermore, the distribution of the JBG revert is currently unknown and without the ability to identify these varieties through morphology, some cogongrass infestations may be the result of JBG reverts. Unfortunately, current molecular methods of identification typically rely on AFLP (Amplified Fragment Length Polymorphisms) and DNA sequencing, both of which are time consuming and costly. Here, we present the first cost-effective and reliable PCR-based molecular genotyping method to accurately distinguish between cogongrass and JBG revert.

A PCR-based Genotyping Method to Distinguish Between Wild-type and Ornamental Varieties of Imperata cylindrica

We provide a cost-effective and rapid molecular genotyping protocol that employs variety-specific PCR primers that target DNA sequence differences within the chloroplast trnL-F spacer region to differentiate between varieties of Imperata cylindrica (cogongrass) that cannot be distinguished by morphology alone. These varieties include the federally listed noxious weed, cogongrass and closely-related, wide-spread ornamental variety, I. cylindrica var. koenigii (Japanese blood grass).

Una basada en PCR genotipificación método para distinguir entre las variedades de tipo silvestre y ornamental de Imperata cylindrica</em

Wild-type I. cylindrica (cogongrass) is one of the top ten worst invasive plants in the world, negatively impacting agricultural and natural resources in ...

Recombineering Homologous Recombination Constructs in Drosophila

The continued development of techniques for fast, large-scale manipulation of endogenous gene loci will broaden the use of Drosophila melanogaster as a genetic model organism for human-disease related research. Recent years have seen technical advancements like homologous recombination and recombineering. However, generating unequivocal null mutations or tagging endogenous proteins remains a substantial effort for most genes. Here, we describe and demonstrate techniques for using recombineering-based cloning methods to generate vectors that can be used to target and manipulate endogenous loci in vivo. Specifically, we have established a combination of three technologies: (1) BAC transgenesis/recombineering, (2) ends-out homologous recombination and (3) Gateway technology to provide a robust, efficient and flexible method for manipulating endogenous genomic loci. In this protocol, we provide step-by-step details about how to (1) design individual vectors, (2) how to clone large fragments of genomic DNA into the homologous recombination vector using gap repair, and (3) how to replace or tag genes of interest within these vectors using a second round of recombineering. Finally, we will also provide a protocol for how to mobilize these cassettes in vivo to generate a knockout, or a tagged gene via knock-in. These methods can easily be adopted for multiple targets in parallel and provide a means for manipulating the Drosophila genome in a timely and efficient manner.

Recombineering Homologous Recombination Constructs in Drosophila

Homologous recombination techniques greatly advance Drosophila genetics by enabling the creation of molecularly precise mutations. The recent adoption of recombineering allows one to manipulate large pieces of DNA and transform them into Drosophila6. The methods presented here combine these techniques to rapidly generate large homologous recombination vectors.

Recombinería homólogos Construcciones recombinación en<em&gt; Drosophila</em

The continued development of techniques for fast, large-scale manipulation of endogenous gene loci will broaden the use of Drosophila melanogaster as a ...

Measuring the Effects of Bacteria on C. Elegans Behavior Using an Egg Retention Assay

C. elegans egg-laying behavior is affected by environmental cues such as osmolarity1 and vibration2. In the total absence of food C. elegans also cease egg-laying and retain fertilized eggs in their uterus3. However, the effect of different sources of food, especially pathogenic bacteria and particularly Enterococcus faecalis, on egg-laying
	behavior is not well characterized. The egg-in-worm (EIW) assay is a useful tool to quantify the effects of different types of bacteria, in this case E. faecalis, on egg- laying behavior.
	
	EIW assays involve counting the number of eggs retained in the uterus of C. elegans4. The EIW assay involves bleaching staged, gravid adult C. elegans to remove the cuticle and separate the retained eggs from the animal. Prior to bleaching, worms are exposed to bacteria (or any type of environmental cue) for a fixed period of time. After bleaching, one is very easily able to count the number of eggs retained inside the uterus of the worms. In this assay, a quantifiable increase in egg retention after E. faecalis exposure can be easily measured. The EIW assay is a behavioral assay that may be used to screen for potentially pathogenic bacteria or the presence of environmental toxins. In addition, the EIW assay may be a tool to screen for drugs that affect neurotransmitter signaling since egg-laying behavior is modulated by neurotransmitters such as serotonin and acetylcholine5-9.

Measuring the Effects of Bacteria on C. Elegans Behavior Using an Egg Retention Assay

An egg-in-worm (EIW) assay is a useful method to quantify egg-laying behavior. Alterations in egg laying can be a behavioral response of the model organism Caenorhabditis elegans to potentially harmful environmental substances such as those produced by pathogenic bacteria.

Medición de los efectos de las bacterias en<em&gt; C. Elegans</em&gt; Comportamiento El uso de un ensayo de retención de huevo

C. elegans egg-laying behavior is  affected by environmental cues such as osmolarity1 and  vibration2. In the total absence of food C. elegans also cease ...

gDNA Enrichment by a Transposase-based Technology for NGS Analysis of the Whole Sequence of BRCA1, BRCA2, and 9 Genes Involved in DNA Damage Repair

The widespread use of Next Generation Sequencing has opened up new avenues for cancer research and diagnosis. NGS will bring huge amounts of new data on cancer, and especially cancer genetics. Current knowledge and future discoveries will make it necessary to study a huge number of genes that could be involved in a genetic predisposition to cancer. In this regard, we developed a Nextera design to study 11 complete genes involved in DNA damage repair. This protocol was developed to safely study 11 genes (ATM, BARD1, BRCA1, BRCA2, BRIP1, CHEK2, PALB2, RAD50, RAD51C, RAD80, and TP53) from promoter to 3&#39;-UTR in 24 patients simultaneously. This protocol, based on transposase technology and gDNA enrichment, gives a great advantage in terms of time for the genetic diagnosis thanks to sample multiplexing. This protocol can be safely used with blood gDNA.

gDNA Enrichment by a Transposase-based Technology for NGS Analysis of the Whole Sequence of BRCA1, BRCA2, and 9 Genes Involved in DNA Damage Repair

gDNA enrichment for NGS sequencing is an easy and powerful tool for the study of constitutional mutations. In this article, we present the procedure to analyse simply the complete sequence of 11 genes involved in DNA damage repair.

gDNA Enriquecimiento por una tecnología basada en la transposasa de Análisis NGS de la secuencia entera de<em&gt; BRCA1</em&gt;,<em&gt; BRCA2</em&gt;, y 9 genes involucrados en la reparación del ADN daños

The widespread use of Next Generation Sequencing has opened up new avenues for cancer research and diagnosis. NGS will bring huge amounts of new data on ...

Identifying Protein-protein Interaction Sites Using Peptide Arrays

Protein-protein interactions mediate most of the processes in the living cell and control homeostasis of the organism. Impaired protein interactions may result in disease, making protein interactions important drug targets. It is thus highly important to understand these interactions at the molecular level. Protein interactions are studied using a variety of techniques ranging from cellular and biochemical assays to quantitative biophysical assays, and these may be performed either with full-length proteins, with protein domains or with peptides. Peptides serve as excellent tools to study protein interactions since peptides can be easily synthesized and allow the focusing on specific interaction sites. Peptide arrays enable the identification of the interaction sites between two proteins as well as screening for peptides that bind the target protein for therapeutic purposes. They also allow high throughput SAR studies. For identification of binding sites, a typical peptide array usually contains partly overlapping 10-20 residues peptides derived from the full sequences of one or more partner proteins of the desired target protein. Screening the array for binding the target protein reveals the binding peptides, corresponding to the binding sites in the partner proteins, in an easy and fast method using only small amount of protein.
In this article we describe a protocol for screening peptide arrays for mapping the interaction sites between a target protein and its partners. The peptide array is designed based on the sequences of the partner proteins taking into account their secondary structures. The arrays used in this protocol were Celluspots arrays prepared by INTAVIS Bioanalytical Instruments. The array is blocked to prevent unspecific binding and then incubated with the studied protein. Detection using an antibody reveals the binding peptides corresponding to the specific interaction sites between the proteins.

Peptide array screening is a high throughput assay for identifying protein-protein interaction sites. This allows mapping multiple interactions of a target protein and can serve as a method for identifying sites for inhibitors that target a protein. Here we describe a protocol for screening and analyzing peptide arrays.

La identificación de sitios de interacción proteína-proteína utilizando péptido arrays

Protein-protein interactions mediate most of the processes in the living cell and control homeostasis of the organism. Impaired protein interactions may ...

Determination of Sialic Acids in Liver and Milk Samples of Wild-type and CMAH Knock-out Mice.

CMAH (cytidine monophosphate-N-acetylneuraminic acid hydroxylase) is responsible for the oxidation of cytidine monophosphate-N-acetylneuraminic acids in mammals. However, humans cannot oxidize cytidine monophosphate-N-acetylneuraminic acid to cytidine monophosphate-N-glycolylneuraminic acid due to a primary exon deletion of the CMAH gene. To understand the effects and implications of the lack of CMAH activity in more detail, a Cmah knock-out model in mice is of keen interest in basic and applied research. The analysis method to determine the phenotype of this mouse model is herein described in detail, and is based on the detection of both N-acetylneuraminic acid and N-glycolylenuraminic acid in the liver and milk of wild-type and Cmah knock-out mice. Endogenous sialic acids are released and derivatized with o-phenylenediamine to generate fluorogenic derivatives, which can be subsequently analyzed by HPLC. The presented protocol can be also applied for the analysis of milk and tissue samples from various other origins, and may be of use to investigate the nutritional and health effects of N-glycolylneuraminic acid.

Determination of Sialic Acids in Liver and Milk Samples of Wild-type and CMAH Knock-out Mice.

We describe a HPLC-based method for the determination of N-acetylneuraminic acid and N-glycolylenuraminic acid in mouse liver and milk.

Determinación de ácidos siálicos en hígado y muestras de leche de tipo salvaje y<em&gt; CMAH</em&gt; Ratones knock-out.

CMAH (cytidine monophosphate-N-acetylneuraminic acid hydroxylase) is responsible for the oxidation of cytidine monophosphate-N-acetylneuraminic acids in ...

A Method to Assess Bacteriocin Effects on the Gut Microbiota of Mice

Very intriguing questions arise with our advancing knowledge on gut microbiota composition and the relationship with health, particularly relating to the factors that contribute to maintaining the population balance. However, there are limited available methodologies to evaluate these factors. Bacteriocins are antimicrobial peptides produced by many bacteria that may confer a competitive advantage for food acquisition and/or niche establishment. Many probiotic lactic acid bacteria (LAB) strains have great potential to promote human and animal health by preventing the growth of pathogens. They can also be used for immuno-modulation, as they produce bacteriocins. However, the antagonistic activity of bacteriocins is normally determined by laboratory bioassays under well-defined but over-simplified conditions compared to the complex gut environment in humans and animals, where bacteria face multifactorial influences from the host and hundreds of microbial species sharing the same niche. This work describes a complete and efficient procedure to assess the effect of a variety of bacteriocins with different target specificities in a murine system. Changes in the microbiota composition during the bacteriocin treatment are monitored using compositional 16S rDNA sequencing. Our approach uses both the bacteriocin producers and their isogenic non-bacteriocin-producing mutants, the latter giving the ability to distinguish bacteriocin-related from non-bacteriocin-related modifications of the microbiota. The fecal DNA extraction and 16S rDNA sequencing methods are consistent and, together with the bioinformatics, constitute a powerful procedure to find faint changes in the bacterial profiles and to establish correlations, in terms of cholesterol and triglyceride concentration, between bacterial populations and health markers. Our protocol is generic and can thus be used to study other compounds or nutrients with the potential to alter the host microbiota composition, either when studying toxicity or beneficial effects.

Bacteriocins are believed to play a key role in defining microbial diversity in different ecological niches. Here, we describe an efficient procedure to assess how bacteriocins affect gut microbiota composition in an animal model.

Un método para evaluar los efectos de bacteriocinas en la microbiota intestinal de ratones

Very intriguing questions arise with our advancing knowledge on gut microbiota composition and the relationship with health, particularly relating to the ...

Investigating the Detrimental Effects of Low Pressure Plasma Sterilization on the Survival of Bacillus subtilis Spores Using Live Cell Microscopy

Plasma sterilization is a promising alternative to conventional sterilization methods for industrial, clinical, and spaceflight purposes. Low pressure plasma (LPP) discharges contain a broad spectrum of active species, which lead to rapid microbial inactivation. To study the efficiency and mechanisms of sterilization by LPP, we use spores of the test organism Bacillus subtilis because of their extraordinary resistance against conventional sterilization procedures. We describe the production of B. subtilis spore monolayers, the sterilization process by low pressure plasma in a double inductively coupled plasma reactor, the characterization of spore morphology using scanning electron microscopy (SEM), and the analysis of germination and outgrowth of spores by live cell microscopy. A major target of plasma species is genomic material (DNA) and repair of plasma-induced DNA lesions upon spore revival is crucial for survival of the organism. Here, we study the germination capacity of spores and the role of DNA repair during spore germination and outgrowth after treatment with LPP by tracking fluorescently-labelled DNA repair proteins (RecA) with time-resolved confocal fluorescence microscopy. Treated and untreated spore monolayers are activated for germination and visualized with an inverted confocal live cell microscope over time to follow the reaction of individual spores. Our observations reveal that the fraction of germinating and outgrowing spores is dependent on the duration of LPP-treatment reaching a minimum after 120 s. RecA-YFP (yellow fluorescence protein) fluorescence was detected only in few spores and developed in all outgrowing cells with a slight elevation in LPP-treated spores. Moreover, some of the vegetative bacteria derived from LPP-treated spores showed an increase in cytoplasm and tended to lyse. The described methods for analysis of individual spores could be exemplary for the study of other aspects of spore germination and outgrowth.

Investigating the Detrimental Effects of Low Pressure Plasma Sterilization on the Survival of Bacillus subtilis Spores Using Live Cell Microscopy

This protocol illustrates the important consecutive steps required to assess the relevance of monitoring vitality parameter and DNA repair processes in reviving Bacillus subtilis spores after treatment with low pressure plasma by tracking fluorescence-labelled DNA repair proteins via time-resolved confocal microscopy and scanning electron microscopy.

Investigar los efectos perjudiciales de baja presión Plasma esterilización sobre la supervivencia de vivir el Bacillus subtilis esporas usando el celular microscopia

Plasma sterilization is a promising alternative to conventional sterilization methods for industrial, clinical, and spaceflight purposes. Low pressure ...