Estamos muy agradecidos a Nancy Papalopulu y Boyan Bonev de asistencia con la electroporación Xenopus. También queremos agradecer a Marc Dionne para la lectura crítica, Green y Jeremy John Wallingford útil para los debates y los miembros del laboratorio de Liu por su apoyo. Este trabajo fue financiado por becas de la BBSRC (BB/E013872/1) y el Wellcome Trust (081880/Z/06/Z) para KJL.

Parte 1A. Equipo Microscopio: alcance vertical estéreo disección con el objetivo de baja potencia <ul><li> Tensión / generador de impulsos: BTX 830 ECM sistema de electroporación de onda cuadrada </li><li> Pipeta extractor: P-87 Micropipeta Extractor (Compañía Sutter Instrument, CA) </li><li> Manipulador: grueso, o combinarse gruesa y fina en función de la preparación. </li><li> Titular de la micropipeta: Herramientas de Bellas Ciencia </li><li> Electrodo: hechos en casa </li><li> Electroporación de cámara: en casa </li></ul> Electrodos en forma de L: <ol><li> Cortar 8 cm de alta pureza 0,4 mm de alambre de tungsteno (Goodfellow) y colocará en el punto medio de una jeringa de 1 ml con masilla (usamos Blu-Tack). Deja 4 cm de alambre de tungsteno expuestos desde la punta de la jeringa y la punta curva en forma de L, de 1 cm desde el extremo (Fig. 1A). </li><li> Recortar la punta para que el extremo mide 0,5 mm de longitud. Tsu punta es la terminal del electrodo. </li><li> En paralelo el exceso de alambre de tungsteno a la jeringa y la utilizan para conectar el generador de impulsos del electrodo. </li><li> Repita el proceso de hacer un par de electrodos. </li><li> Conecte los electrodos al generador de pulso cuadrado de onda a través de cables de CC. </li></ol> Electroporación cámara <ol><li> Línea inferior de 90 mm plato con ~ 5 mm de plastilina no tóxica. </li><li> Rellene plato con los medios de comunicación de la electroporación. </li><li> Usando N º 5 pinzas de relojero hacerse un bien en forma de T (Fig. 2). El bien siempre debe medir ~ 2 mm x 2 mm x 10 mm y el corto ~ 2 mm x 2 mm x 5 mm. El plato de electroporación se pueden lavar y reutilizar. </li></ol> Parte 1B. Reactivos ADN o macromoléculas cargadas <ul><li> Micropipetas: 1 mm de ancho, 4 &quot;de largo capilares de vidrio de borosilicato (WPI, TW100-F) </li><li> Los medios de cultivo: Normal Medios Anfibios (NAM) </li><li> &gt; 10 X stock: 1100 mM NaCl, 20mM KCl, 10 mM de Ca (NO 3) 2 • 4H 2 O, 1 mM EDTA. <ul><li> Autoclave y almacenar a 4 ° C. </li><li> 1 X NAM: Diluir 10 veces desde el almacén de amortiguación, con 0,1 mM NaHCO 3 y 0,2 mM Na 3 PO 4. </li></ul></li><li> Renacuajos Xenopus laevis, etapa 28 </li></ul> Preparación del ADN: <ol><li> Prepare plásmidos de expresión a través de protocolos estándar. </li><li> Resuspender el ADN a una concentración final de 1 mg / l en la nucleasa libre de H 2 0. </li></ol> * Hemos tenido éxito con vectores que contienen un fuerte promotor de CMV, como pCS2 + [16]. Para el análisis de linaje, por lo general incluyen el ADN que codifica la proteína verde fluorescente (GFP pCS2 +) a una concentración final de 0,1 mg / l. Concentraciones [de ADN entre 0.1-3 mg / l también fueron probados. Se encontró que las concentraciones por debajo de 0,8 mg / l células ineficiente etiqueta, mientras que las concentraciones de ADN de más de 2 mg / m yu, l no ha mejorado la eficiencia de la electroporación]. Morfolino oligonucleótidos preparación: (Nota:. OM deben ser fluoresceinado (3&#39;-carboxifluoresceína modificados) o acusado de otro tipo) <ol><li> Resuspender morfolino oligonucleótidos (OM) (GeneTools, <a href="http://www.genetools.com" target="_blank">www.genetools.com</a> ) a una concentración de 2 mM de nucleasa libre de H 2 0. </li><li> Alícuota de la solución madre de calor a 65 ° C durante 5 minutos. </li><li> Se diluye a la concentración final de 0,5 mM en agua libre de nucleasa. </li></ol> * 0.1-1mm soluciones MO fueron probados. 0,5 mM soluciones MO fueron suficientes para la electroporación de muchas células mesenquimales. Micropipetas <ol><li> Prepare micropipetas de vidrio de borosilicato capilares (1 mm de ancho, 4 &quot;de largo, no WPI. TW100-F). Utilice extractor de aguja para preparar micropipetas con un cono 8-12 mm de largo y de punta fina. </li><li> Aplastar a punta de ~ 2 mmde la punta con las pinzas, la creación de una ruptura irregulares. </li></ol> Medios de comunicación <ol><li> Medio de incubación: Preparar fresca 3.4 normal de Medios Anfibios (NAM) de 1x de valores. Añadir 0,025 mg / ml de gentamicina. </li><li> Los medios de comunicación de la electroporación: como el anterior, con el 0,1% de benzocaína (Sigma, 06950). </li></ol> 2. Electroporación Micropipeta de configuración <ol><li> Rellene micropipeta con una solución inyectable ~ l. </li><li> Seguro micropipeta en micromanipulador y se unen a microinyector (Picospritzer II). </li><li> Micropipeta ángulo a 50 ° de la mesa. </li><li> Ajuste la presión de inyección a 20 PSI. </li><li> Calibrar micropipeta para inyectar 30 nl por impulso. </li></ol> Tadpole preparación <ol><li> Anestesiar etapa 28 larvas de Xenopus incubando en los medios de comunicación de la electroporación por 5 minutos. </li><li> Transferencia de renacuajo anestesiado en la cámara de electroporación llena de medios de comunicación de la electroporación. La posición del embriónen el tiempo y modo que la cabeza se apoya en el cruce con la cara dorsal hacia abajo y la parte ventral expuesta. La cabeza debe estar ligeramente elevada en comparación con la cola. </li><li> Con unas pinzas, suavemente seguro renacuajo bien en los alrededores de plastilina. (Nota:.. Si el renacuajo no está asegurada, puede contracción y contacto de los electrodos durante la electroporación En este caso, descartar el renacuajo como pañuelos de papel se verá seriamente dañada) </li></ol> Electroporación <ol><li> Inserte la punta de micropipeta inmediatamente posterior a la glándula de cemento y en mesénquima facial. </li><li> Inyectar la solución de 30 nl en mesénquima. </li><li> Retraer micropipeta. </li><li> Alinear rápidamente puntas de los electrodos paralelos a la cabeza del embrión (Fig. 3). </li><li> Aplicar 8 50 ms, legumbres 20mV cuadrados. </li><li> Retraer los electrodos. </li><li> El uso de pinzas cuidadosamente liberación renacuajo de bien y de transferencia de hasta 3 / 4 NAM, 0,025 mg / ml de gentamicina. </li><li> Los renacuajos pueden ser incubados en 3 / 4 NAM, 0,025 mg / ml Overnight, o más. </li><li> Seleccionar embriones para la electroporación eficiente mediante microscopía de fluorescencia después de 24 horas. </li></ol> 3. Los resultados representativos: El uso de moléculas fluorescentes permite la detección fácil de embriones electroporated. La figura 4 muestra un lote típico de MO electroporated renacuajos ~ 12, 48 y 96 horas después de la electroporación, se incubaron a 14,5 ° C. Mediante microscopía de fluorescencia, OM se puede visualizar inmediatamente después de la electroporación y persistir durante varios días después de la electroporación. En nuestra experiencia, la fluorescencia es débilmente evidente en la etapa 46 (~ 5 días después). En los cartílagos, la fluorescencia disminuye dramáticamente después de la aparición de la diferenciación (~ º 42), sin embargo, MO fluorescencia persiste con más fuerza en otros tipos celulares, como el endodermo faríngeo. Microscopía de fluorescencia muestra que los oligonucleótidos se incorporan en varios tejidos craneofaciales incluyendo el cartílago. Oligonucleótidos c fluorescenciaa menudo un ser visualizado en el tejido en ambos lados de la cabeza. Esto es probablemente debido a la rápida difusión de la solución inyectable en todo el mesénquima craneofacial sueltos antes de la electroporación. <img alt="Figura 1" src="/files/ftp_upload/3381/3381fig1.jpg" /> Figura 1 electrodos hechos en casa. En forma de L de alambre de tungsteno es conectada a una jeringa de 1 ml con no-tóxico de barro o masilla. (A) El término del electrodo mide 5 mm. (B) Coloque un par de electrodos, de modo que los extremos son paralelas. Electrodos se conectan al generador de impulsos a través de cables de CC. <img alt="Figura 2" src="/files/ftp_upload/3381/3381fig2.jpg" /> Figura 2 cámara de electroporación. (A) 90 mm plato forrado con plastilina está lleno de medios de comunicación y una cámara en forma de T tallada con fórceps No relojero de 5 años. (B) El lado más largo mide 2 mm x 2 mm X10 mmmientras que las medidas sólo 2 mm x 2 mm x 5 mm. La cabeza del embrión se basa en el cruce, la parte ventral hacia arriba. <img alt="Figura 3" src="/files/ftp_upload/3381/3381fig3.jpg" /> Figura 3 Esquema que ilustra el procedimiento de electroporación. St. 28 renacuajo se coloca en la cámara de electroporación, lado ventral hacia arriba. Micropipeta se inserta en la glándula de cemento facial mesénquima subyacente. Inyectar. Micropipeta se elimina en forma de L y los electrodos están alineados en paralelo que flanquean la cabeza. Aplicar ocho de 50 ms, 20 mV pulsos cuadrados. Retraer los electrodos. Renacuajos crecen en etapas deseado. Visualizar OM o la expresión de GFP mediante microscopía de fluorescencia. <img alt="Figura 4" src="/files/ftp_upload/3381/3381fig4.jpg" /> Figura 4 renacuajos Representante 12 (A), 48 (B) y 96 (C) horas después de la electroporación (etapas 30, 34 y 44, respectivamente). (A &quot;B&quot;) MO fluorescente se puede visualizar en mesénquima craneofacial en el ciervoes 30 y 34. La fluorescencia se puede detectar en los cartílagos en la etapa 44 (punta de flecha, C &#39;-C &quot;). El intestino es muy autofluorescentes.

<ol>
	<li>Bonev, B., Pisco, A., Papalopulu, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MicroRNA-9+reveals+regional+diversity+of+neural+progenitors+along+the+anterior-posterior+axis.">MicroRNA-9 reveals regional diversity of neural progenitors along the anterior-posterior axis.</a> Dev. Cell. 20, 19-32 (2011).</li><li>Haas, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-cell+electroporation+for+gene+transfer+in+vivo.">Single-cell electroporation for gene transfer in vivo.</a> Neuron. 29, 583-591 (2001).</li><li>Calegari, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue-specific+RNA+interference+in+post-implantation+mouse+embryos+using+directional+electroporation+and+whole+embryo+culture.">Tissue-specific RNA interference in post-implantation mouse embryos using directional electroporation and whole embryo culture.</a> Differentiation. 72, 92-102 (2004).</li><li>Drinjakovic, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=E3+ligase+Nedd4+promotes+axon+branching+by+downregulating+PTEN.">E3 ligase Nedd4 promotes axon branching by downregulating PTEN.</a> Neuron. 65, 341-357 (2010).</li><li>Falk, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electroporation+of+cDNA/Morpholinos+to+targeted+areas+of+embryonic+CNS+in+Xenopus.">Electroporation of cDNA/Morpholinos to targeted areas of embryonic CNS in Xenopus.</a> BMC. Dev. Biol. 7, 107-107 (2007).</li><li>Hewapathirane, D. S., Haas, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single+Cell+Electroporation+in+vivo+within+the+Intact+Developing+Brain.">Single Cell Electroporation in vivo within the Intact Developing Brain.</a> J. Vis. Exp. (17), e705-e705 (2008).</li><li>Kuriyama, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tsukushi+controls+ectodermal+patterning+and+neural+crest+specification+in+Xenopus+by+direct+regulation.">Tsukushi controls ectodermal patterning and neural crest specification in Xenopus by direct regulation.</a> of BMP4 and X-delta-1 activity. Development. 133, 75-88 (2006).</li><li>Mende, M., Christophorou, N. A., Streit, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Specific+and+effective+gene+knock-down+in+early+chick+embryos+using+morpholinos+but+not+pRFPRNAi+vectors.">Specific and effective gene knock-down in early chick embryos using morpholinos but not pRFPRNAi vectors.</a> Mech. Dev. 125, 947-962 (2008).</li><li>Neumann, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+transfer+into+mouse+lyoma+cells+by+electroporation+in+high+electric+fields.">Gene transfer into mouse lyoma cells by electroporation in high electric fields.</a> Embo. J. 1, 841-845 (1982).</li><li>Price, S. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+motor+neuron+pool+sorting+by+differential+expression+of+type+II+cadherins.">Regulation of motor neuron pool sorting by differential expression of type II cadherins.</a> Cell. 109, 205-216 (2002).</li><li>Grossin, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+gene+transfer+into+rat+articular+cartilage+by+in+vivo+electroporation.">Direct gene transfer into rat articular cartilage by in vivo electroporation.</a> Faseb. J. 17, 829-835 (2003).</li><li>Khoury, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+comparative+study+on+intra-articular+versus+systemic+gene+electrotransfer+in+experimental+arthritis.">A comparative study on intra-articular versus systemic gene electrotransfer in experimental arthritis.</a> J. Gene. Med. 8, 1027-1036 (2006).</li><li>Takahashi, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Down-regulation+of+cathepsin+K+in+synovium+leads+to+progression+of+osteoarthritis+in+rabbits.">Down-regulation of cathepsin K in synovium leads to progression of osteoarthritis in rabbits.</a> Arthritis. Rheum. 60, 2372-2380 (2009).</li><li>Sive, H. L., Grainger, R. M., Harland, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Early Development of Xenopus laevis: A Laboratory Manual. , (2000).</li><li>Wheeler, G. N., Brandli, A. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simple+vertebrate+models+for+chemical+genetics+and+drug+discovery+screens:+lessons+from+zebrafish+and+Xenopus.">Simple vertebrate models for chemical genetics and drug discovery screens: lessons from zebrafish and Xenopus.</a> Dev. Dyn. 238, 1287-1308 (2009).</li><li>Turner, D. L., Weintraub, H. <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+achaete-scute+homolog+3+in+Xenopus+embryos+converts+ectodermal+cells+to+a+neural+fate.">Expression of achaete-scute homolog 3 in Xenopus embryos converts ectodermal cells to a neural fate.</a> Genes. Dev. 8, 1434-1447 (1994).</li></ol>

Los autores no tienen conflicto de intereses.

En este vídeo, hemos demostrado la viabilidad de la entrega de genes mediada por electroporación en el mesénquima facial de los renacuajos Xenopus. El uso de este enfoque, podemos evitar los primeros efectos del desarrollo de la manipulación de la función del gen que nos permite apuntar tejidos específicos en momentos posteriores. Nuestros estudios muestran que la población heterogénea de células mesenquimales craneofacial puede verse afectada, lo que nos permite estudiar el linaje de las células por electroporación, así como los requisitos de la célula autónoma de las proteínas de interés. Combinado con imágenes en vivo, se puede utilizar este método para estudiar la función génica, con el tiempo, durante el desarrollo craneofacial. Este nuevo método pone de manifiesto la maleabilidad de Xenopus para el estudio de la organogénesis. Creemos que este método puede ser adaptado ampliamente para estudiar la morfogénesis y la diferenciación de otros tejidos.

A correction was made to <a href="http://http://www.jove.com/video/3381">Electroporation of Craniofacial Mesenchyme</a>. There was an incorrect unit used for the voltage.

Erratum: Electroporation of Craniofacial Mesenchyme

electroporation of craniofacial mesenchyme

La electroporación es un método eficaz para el suministro de ADN y otras macromoléculas cargadas en los tejidos en los puntos precisos de tiempo y en lugares precisos. Por ejemplo, la electroporación se ha utilizado con gran éxito para estudiar el desarrollo neuronal y de la retina en Xenopus, pollo y ratón 1.10. Sin embargo, es importante señalar que en todos estos estudios, los investigadores no estaban dirigidas a los tejidos blandos. Debido a que estamos interesados ​​en el desarrollo craneofacial, adaptamos un método para apuntar mesénquima facial. Cuando se realizaron búsquedas en la literatura, encontramos, para nuestra sorpresa, muy pocos informes de la transferencia de genes con éxito en el tejido cartilaginoso. La mayoría de estos estudios fueron estudios de terapia génica, como la entrega de siRNA o de proteínas en líneas celulares condrogénica, o, en modelos animales de artritis 11-13. En otros sistemas, como el pollo o el ratón, la electroporación de mesénquima facial ha sido un desafío (COMUNICACI personaliones, Departamento de Desarrollo Craneofacial, KCL). La hipótesis de que la electroporación en los tejidos cartilaginosos y procartilaginous en Xenopus podría funcionar mejor. En nuestros estudios, nos muestran que la transferencia de genes en los cartílagos faciales se produce de manera eficiente en las etapas iniciales (28), cuando el primordio facial sigue siendo compuesto de los tejidos blandos antes de la diferenciación del cartílago. Xenopus es un sistema de vertebrados muy accesible para el análisis del desarrollo craneofacial. Estructuras craneofaciales son más visibles en Xenopus que en cualquier modelo de otros vertebrados, principalmente debido a embriones de Xenopus son fertilizados externamente, lo que permite el análisis de las etapas más tempranas, y facilitar imágenes en vivo a una resolución única célula, así como la reutilización de las 14 madres. Entre los modelos de vertebrados desarrollo externo, Xenopus es más útil para el análisis craneofaciales que el pez cebra, Xenopus, como las larvas son más grandes y más fáciles de dissect, y el desarrollo de la región facial es más accesible a las imágenes de la región equivalente en pescado. Además, Xenopus es evolutivamente más cerca de los humanos que el pez cebra (~ 100 millones años más) 15. Por último, en estas etapas, los renacuajos Xenopus son transparentes, y la expresión simultánea de proteínas fluorescentes o moléculas que permiten una fácil visualización de los cartílagos en desarrollo. Creemos que este enfoque nos permitirá poner a prueba rápida y eficiente las moléculas candidatas en un sistema modelo in vivo.

Watch this Scientific Journal Video about Electroporation of Craniofacial Mesenchyme at JoVE.com

Electroporation of Craniofacial Mesenchyme

Cartílagos craneofaciales en estrecho contacto con otros tejidos, y son difíciles de manipular en animales vivos. Estamos utilizando electroporación para ofrecer herramientas moleculares durante el crecimiento del esqueleto craneofacial sin pasar por los primeros efectos de embriones. Este enfoque nos permite analizar de manera eficiente las moléculas candidatas<em&gt; In vivo.</em

Electroporación de mesénquima craneofacial

Electroporation is an efficient method of delivering DNA and other charged macromolecules into tissues at precise time points and in precise locations. ...

electroporation-of-craniofacial-mesenchyme

Research

JoVE Journal

Biology

11.8K vistas.  King's College London. Cartílagos craneofaciales en estrecho contacto con otros tejidos, y son difíciles de manipular en animales vivos. Estamos utilizando electroporación para ofrecer herramientas moleculares durante el crecimiento del esqueleto craneofacial sin pasar por los primeros efectos de embriones. Este enfoque nos permite analizar de manera eficiente las moléculas candidatas In vivo.

Video: Electroporation of Craniofacial Mesenchyme

Electroporation of Mycobacteria

High efficiency transformation is a major limitation in the study of mycobacteria. The genus Mycobacterium can be difficult to transform; this is mainly caused by the thick and waxy cell wall, but is compounded by the fact that most molecular techniques have been developed for distantly-related species such as Escherichia coli and Bacillus subtilis. In spite of these obstacles, mycobacterial plasmids have been identified and DNA transformation of many mycobacterial species have now been described. The most successful method for introducing DNA into mycobacteria is electroporation. Many parameters contribute to successful transformation; these include the species/strain, the nature of the transforming DNA, the selectable marker used, the growth medium, and the  conditions for the electroporation pulse. Optimized methods for the transformation of both slow- and fast-grower are detailed here. Transformation efficiencies for different mycobacterial species and with various selectable markers are reported.

Mycobacterial pathogenic strategies remain poorly understood. The slow growth rate of most species, the impenetrable nature of the cell-wall, and the hazards of working with pathogens make mycobacteria difficult to study and are largely responsible for our poor understanding of these organisms. In this video we will demonstrate the technique of electroporation, which involves subjecting cells to a brief high electrical impulse to allow the entry of DNA. It is the most widely used method for introducing DNA into mycobacterial cells.

Electroporación de micobacterias

High efficiency transformation is a major limitation in the study of mycobacteria. The genus Mycobacterium can be difficult to transform; this is mainly ...

Investigación

Biología

Antibody Profiling by Luciferase Immunoprecipitation Systems (LIPS)

Technologies for comprehensively understanding and quantifying antibody profiles to autoantigens and infectious agents may yield new insights into disease mechanisms and may elucidate new markers to substratify disease with different clinical features and better understand pathogenesis. We have developed a highly quantitative method called Luciferase Immunoprecipitation Systems (LIPS) for profiling patient sera antibody responses to autoantigens and pathogen antigens associated with infection. Unlike ELISAs, the highly sensitive LIPS is easily implemented to survey humoral serological response profiles to different antigens in a universal format and produces dynamic antibody titer ranges up to 6 log10 for some antigens. In these studies, quantitative profiling by LIPS of patient humoral responses against panels of antigens or even the entire proteome of some pathogens (i.e. HIV), is typically more informative than testing a single antigen by ELISA. In addition, LIPS also eliminates time and effort needed to produce highly purified antigens as well as the labor-intensive assay optimization steps needed for standard ELISAs. Here we provide a detailed protocol describing the technical aspects of performing LIPS assays for readily profiling antibody responses to single or multiple antigens.

Antibody Profiling by Luciferase Immunoprecipitation Systems LIPS

The technical aspects of performing LIPS (Luciferase Immunoprecipitation Systems) are described. The overall approach involves expressing chimeric genes encoding antigens fused to Renilla luciferase (Ruc) in mammalian cells. Crude Ruc-antigen extracts are then prepared and, without purification, employed in immunoprecipitation assays to quantify antibodies.

Perfiles de anticuerpos por los sistemas de inmunoprecipitación luciferasa (LIPS)

Technologies for comprehensively understanding and quantifying antibody profiles to autoantigens and infectious agents may yield new insights into disease ...

Assessing Signaling Properties of Ectodermal Epithelia During Craniofacial Development

The accessibility of avian embryos has helped experimental embryologists understand the fates of cells during development and the role of tissue interactions that regulate patterning and morphogenesis of vertebrates (e.g., 1, 2, 3, 4). Here, we illustrate a method that exploits this accessibility to test the signaling and patterning properties of ectodermal tissues during facial development. In these experiments, we create quail-chick 5 or mouse-chick 6 chimeras by transplanting the surface cephalic ectoderm that covers the upper jaw from quail or mouse onto either the same region or an ectopic region of chick embryos. The use of quail as donor tissue for transplantation into chicks was developed to take advantage of a nucleolar marker present in quail but not chick cells, thus allowing investigators to distinguish host and donor tissues 7. Similarly, a repetitive element is present in the mouse genome and is expressed ubiquitously, which allows us to distinguish host and donor tissues in mouse-chick chimeras 8. The use of mouse ectoderm as donor tissue will greatly extend our understanding of these tissue interactions, because this will allow us to test the signaling properties of ectoderm derived from various mutant embryos.

This article describes a tissue transplantation technique that was designed to test the signaling and patterning properties of surface cephalic ectoderm during craniofacial development.

La evaluación de las propiedades de señalización de ectodérmica epitelio durante el desarrollo craneofacial

The accessibility of avian embryos has helped experimental embryologists understand the fates of cells during development and the role of tissue ...

An Explant Assay for Assessing Cellular Behavior of the Cranial Mesenchyme

The central nervous system is derived from the neural plate that undergoes a series of complex morphogenetic movements resulting in formation of the neural tube in a process known as neurulation. During neurulation, morphogenesis of the mesenchyme that underlies the neural plate is believed to drive neural fold elevation. The cranial mesenchyme is comprised of the paraxial mesoderm and neural crest cells. The cells of the cranial mesenchyme form a pourous meshwork composed of stellate shaped cells and intermingling extracellular matrix (ECM) strands that support the neural folds. During neurulation, the cranial mesenchyme undergoes stereotypical rearrangements resulting in its expansion and these movements are believed to provide a driving force for neural fold elevation. However, the pathways and cellular behaviors that drive cranial mesenchyme morphogenesis remain poorly studied. Interactions between the ECM and the cells of the cranial mesenchyme underly these cell behaviors. Here we describe a simple ex vivo explant assay devised to characterize the behaviors of these cells. This assay is amendable to pharmacological manipulations to dissect the signaling pathways involved and live imaging analyses to further characterize the behavior of these cells. We present a representative experiment demonstrating the utility of this assay in characterizing the migratory properties of the cranial mesenchyme on a variety of ECM components.

The cranial mesenchyme undergoes dramatic morphogenic movements that likely provides a driving force for elevation of the neural folds1,2. Here we describe a simple ex vivo explant assay to characterize the cellular behaviors of the cranial mesenchyme during neurulation. This assay has numerous applications including being amenable to pharmacological manipulations and live imaging analyses.

Un ensayo de explante para evaluar el comportamiento celular del mesénquima craneal

The central nervous system is derived from the neural plate that undergoes a series of complex morphogenetic movements resulting in formation of the ...

Genome-wide Gene Deletions in Streptococcus sanguinis by High Throughput PCR

Transposon mutagenesis and single-gene deletion are two methods applied in genome-wide gene knockout in bacteria 1,2. Although transposon mutagenesis is less time consuming, less costly, and does not require completed genome information, there are two weaknesses in this method: (1) the possibility of a disparate mutants in the mixed mutant library that counter-selects mutants with decreased competition; and (2) the possibility of partial gene inactivation whereby genes do not entirely lose their function following the insertion of a transposon. Single-gene deletion analysis may compensate for the drawbacks associated with transposon mutagenesis. To improve the efficiency of genome-wide single gene deletion, we attempt to establish a high-throughput technique for genome-wide single gene deletion using Streptococcus sanguinis as a model organism. Each gene deletion construct in S. sanguinis genome is designed to comprise 1-kb upstream of the targeted gene, the aphA-3 gene, encoding kanamycin resistance protein, and 1-kb downstream of the targeted gene. Three sets of primers F1/R1, F2/R2, and F3/R3, respectively, are designed and synthesized in a 96-well plate format for PCR-amplifications of those three components of each deletion construct. Primers R1 and F3 contain 25-bp sequences that are complementary to regions of the aphA-3 gene at their 5' end. A large scale PCR amplification of the aphA-3 gene is performed once for creating all single-gene deletion constructs. The promoter of aphA-3 gene is initially excluded to minimize the potential polar effect of kanamycin cassette. To create the gene deletion constructs, high-throughput PCR amplification and purification are performed in a 96-well plate format. A linear recombinant PCR amplicon for each gene deletion will be made up through four PCR reactions using high-fidelity DNA polymerase. The initial exponential growth phase of S. sanguinis cultured in Todd Hewitt broth supplemented with 2.5% inactivated horse serum is used to increase competence for the transformation of PCR-recombinant constructs. Under this condition, up to 20% of S. sanguinis cells can be transformed using ~50 ng of DNA. Based on this approach, 2,048 mutants with single-gene deletion were ultimately obtained from the 2,270 genes in S. sanguinis excluding four gene ORFs contained entirely within other ORFs in S. sanguinis SK36 and 218 potential essential genes. The technique on creating gene deletion constructs is high throughput and could be easy to use in genome-wide single gene deletions for any transformable bacteria.

Genome-wide Gene Deletions in Streptococcus sanguinis by High Throughput PCR

An efficient genome-wide single gene mutation method has been established using Streptococcus sanguinis as a model organism. This method has achieved via high throughput recombinant PCRs and transformations.

Genoma escala supresiones de genes en<em&gt; Streptococcus sanguinis</em&gt; Por PCR de alto rendimiento

Transposon  mutagenesis and single-gene deletion are two methods applied in genome-wide  gene knockout in bacteria 1,2. Although transposon mutagenesis is ...

Separation of Mouse Embryonic Facial Ectoderm and Mesenchyme

Orofacial clefts are the most frequent craniofacial defects, which affect 1.5 in 1,000 newborns worldwide1,2. Orofacial clefting is caused by abnormal facial development3. In human and mouse, initial growth and patterning of the face relies on several small buds of tissue, the facial prominences4,5. The face is derived from six main prominences: paired frontal nasal processes (FNP), maxillary prominences (MxP) and mandibular prominences (MdP). These prominences consist of swellings of mesenchyme that are encased in an overlying epithelium. Studies in multiple species have shown that signaling crosstalk between facial ectoderm and mesenchyme is critical for shaping the face6. Yet, mechanistic details concerning the genes involved in these signaling relays are lacking. One way to gain a comprehensive understanding of gene expression, transcription factor binding, and chromatin marks associated with the developing facial ectoderm and mesenchyme is to isolate and characterize the separated tissue compartments.
Here we present a method for separating facial ectoderm and mesenchyme at embryonic day (E) 10.5, a critical developmental stage in mouse facial formation that precedes fusion of the prominences. Our method is adapted from the approach we have previously used for dissecting facial prominences7. In this earlier study we had employed inbred C57BL/6 mice as this strain has become a standard for genetics, genomics and facial morphology8. Here, though, due to the more limited quantities of tissue available, we have utilized the outbred CD-1 strain that is cheaper to purchase, more robust for husbandry, and tending to produce more embryos (12-18) per litter than any inbred mouse strain8. Following embryo isolation, neutral protease Dispase II was used to treat the whole embryo. Then, the facial prominences were dissected out, and the facial ectoderm was separated from the mesenchyme. This method keeps both the facial ectoderm and mesenchyme intact. The samples obtained using this methodology can be used for techniques including protein detection, chromatin immunoprecipitation (ChIP) assay, microarray studies, and RNA-seq.

A protocol for separation of embryo facial ectoderm and mesenchyme is described. We use Dispase II to treat whole embryos first, dissect whole facial prominences out, and then separate the facial ectoderm and mesenchyme.

La separación de Ratón Facial ectodermo embrionario y mesénquima

 Orofacial clefts are the most frequent  craniofacial defects, which affect 1.5 in 1,000 newborns worldwide1,2. Orofacial clefting is caused by abnormal ...

Visualization of Craniofacial Development in the sox10: kaede Transgenic Zebrafish Line Using Time-lapse Confocal Microscopy

Vertebrate palatogenesis is a highly choreographed and complex developmental process, which involves migration of cranial neural crest (CNC) cells, convergence and extension of facial prominences, and maturation of the craniofacial skeleton. To study the contribution of the cranial neural crest to specific regions of the zebrafish palate a sox10: kaede transgenic zebrafish line was generated. Sox10 provides lineage restriction of the kaede reporter protein to the neural crest, thereby making the cell labeling a more precise process than traditional dye or reporter mRNA injection. Kaede is a photo-convertible protein that turns from green to red after photo activation and makes it possible to follow cells precisely. The sox10: kaede transgenic line was used to perform lineage analysis to delineate CNC cell populations that give rise to maxillary versus mandibular elements and illustrate homology of facial prominences to amniotes. This protocol describes the steps to generate a live time-lapse video of a sox10: kaede zebrafish embryo. Development of the ethmoid plate will serve as a practical example. This protocol can be applied to making a time-lapse confocal recording of any kaede or similar photoconvertible reporter protein in transgenic zebrafish. Furthermore, it can be used to capture not only normal, but also abnormal development of craniofacial structures in the zebrafish mutants.

Visualization of experimental data has become a key element in presenting results to the scientific community. Generation of live time-lapse recording of growing embryos contributes to better presentation and understanding of complex developmental processes. This protocol is a step-by-step guide to cell labeling via photoconversion of kaede protein in zebrafish.

Visualización de Desarrollo Craneofacial en los SOX10: Kaede pez cebra transgénico Línea Usando Time-lapse microscopía confocal

Vertebrate palatogenesis is a highly choreographed and complex developmental process, which involves migration of cranial neural crest (CNC) cells, ...

Analyzing Craniofacial Morphogenesis in Zebrafish Using 4D Confocal Microscopy

Time-lapse imaging is a technique that allows for the direct observation of the process of morphogenesis, or the generation of shape. Due to their optical clarity and amenability to genetic manipulation, the zebrafish embryo has become a popular model organism with which to perform time-lapse analysis of morphogenesis in living embryos. Confocal imaging of a live zebrafish embryo requires that a tissue of interest is persistently labeled with a fluorescent marker, such as a transgene or injected dye. The process demands that the embryo is anesthetized and held in place in such a way that healthy development proceeds normally. Parameters for imaging must be set to account for three-dimensional growth and to balance the demands of resolving individual cells while getting quick snapshots of development. Our results demonstrate the ability to perform long-term in vivo imaging of fluorescence-labeled zebrafish embryos and to detect varied tissue behaviors in the cranial neural crest that cause craniofacial abnormalities. Developmental delays caused by anesthesia and mounting are minimal, and embryos are unharmed by the process. Time-lapse imaged embryos can be returned to liquid medium and subsequently imaged or fixed at later points in development. With an increasing abundance of transgenic zebrafish lines and well-characterized fate mapping and transplantation techniques, imaging any desired tissue is possible. As such, time-lapse in vivo imaging combines powerfully with zebrafish genetic methods, including analyses of mutant and microinjected embryos.

Time-lapse confocal imaging is a powerful technique useful for characterizing embryonic development. Here, we describe the methodology and characterize craniofacial morphogenesis in wild-type, as well as pdgfra, smad5, and smo mutant embryos.

Analizando Craneofacial en la morfogénesis de pez cebra Utilizando 4D Microscopía Confocal

Time-lapse imaging is a technique that allows for the direct observation of the process of morphogenesis, or the generation of shape. Due to their optical ...

Alternative Cultures for Human Pluripotent Stem Cell Production, Maintenance, and Genetic Analysis

Human pluripotent stem cells (hPSCs) hold great promise for regenerative medicine and biopharmaceutical applications. Currently, optimal culture and efficient expansion of large amounts of clinical-grade hPSCs are critical issues in hPSC-based therapies. Conventionally, hPSCs are propagated as colonies on both feeder and feeder-free culture systems. However, these methods have several major limitations, including low cell yields and generation of heterogeneously differentiated cells. To improve current hPSC culture methods, we have recently developed a new method, which is based on non-colony type monolayer (NCM) culture of dissociated single cells. Here, we present detailed NCM protocols based on the Rho-associated kinase (ROCK) inhibitor Y-27632. We also provide new information regarding NCM culture with different small molecules such as Y-39983 (ROCK I inhibitor), phenylbenzodioxane (ROCK II inhibitor), and thiazovivin (a novel ROCK inhibitor). We further extend our basic protocol to cultivate hPSCs on defined extracellular proteins such as the laminin isoform 521 (LN-521) without the use of ROCK inhibitors. Moreover, based on NCM, we have demonstrated efficient transfection or transduction of plasmid DNAs, lentiviral particles, and oligonucleotide-based microRNAs into hPSCs in order to genetically modify these cells for molecular analyses and drug discovery. The NCM-based methods overcome the major shortcomings of colony-type culture, and thus may be suitable for producing large amounts of homogeneous hPSCs for future clinical therapies, stem cell research, and drug discovery.

Here, we present human pluripotent stem cell (hPSC) culture protocols, based on non-colony type monolayer (NCM) growth of dissociated single cells. This new method, utilizing Rho-associated kinase inhibitors or the laminin isoform 521 (LN-521), is suitable for producing large amounts of homogeneous hPSCs, genetic manipulation, and drug discovery.

Culturas alternativas para Pluripotentes Stem Cell Production, mantenimiento y análisis genético

Human pluripotent stem cells (hPSCs) hold great promise for regenerative medicine and biopharmaceutical applications. Currently, optimal culture and ...

Assessing Species-specific Contributions To Craniofacial Development Using Quail-duck Chimeras

The generation of chimeric embryos is a widespread and powerful approach to study cell fates, tissue interactions, and species-specific contributions to the histological and morphological development of vertebrate embryos. In particular, the use of chimeric embryos has established the importance of neural crest in directing the species-specific morphology of the craniofacial complex. The method described herein utilizes two avian species, duck and quail, with remarkably different craniofacial morphology. This method greatly facilitates the investigation of molecular and cellular regulation of species-specific pattern in the craniofacial complex.&#160;Experiments in quail and duck chimeric embryos have already revealed neural crest-mediated tissue interactions and cell-autonomous behaviors that regulate species-specific pattern in the craniofacial skeleton, musculature, and integument. The great diversity of neural crest derivatives suggests significant potential for future applications of the quail-duck chimeric system to understanding vertebrate development, disease, and evolution.

This article describes a method to generate chimeric embryos that are&#160;designed to test the species-specific contributions of neural crest and/or other tissues to craniofacial development.

Evaluar Aportes específicos de la especie para el desarrollo craneofacial Utilizando quimeras Quail-pato

The generation of chimeric embryos is a widespread and powerful approach to study cell fates, tissue interactions, and species-specific contributions to ...