Los autores desean agradecer al Dr. Peiman Hemmati (Departamento de Medicina de la Universidad de Wisconsin-Madison) para ofrecer generosamente los CSM H1, y el Dr. Tim Hacker, Dr. Gouqing canción y la Sra. Jill Koch, de la Universidad de Wisconsin, la fisiología cardiovascular Fondo central para la realización de cirugías de ratón. Este trabajo fue apoyado por la Fundación Nacional de Ciencia a través de una Beca de Investigación de Postgrado de Brian Freeman y NIH R21 HL089679.

1. Transfección de células de donantes <ol><li> Las células de la cosecha madre mesenquimales (MSC, derivadas de células madre embrionarias H1 donado amablemente por el Dr. Peiman Hematti, alternativamente, cualquier tipo de célula de cualquier especie la hipótesis de que el fusible en vivo podrían ser empleados) cuando el 70 - 80% confluentes con tripsina 1X (Mediatech, Manassas VA) durante 5 min. inactivar la tripsina con α-MEM medio completo (sin antibióticos, Invitrogen, Carlsbad CA) 18. Centrifugar a 300 xg durante 5 min. </li><li> Aspirar el sobrenadante con cuidado y vuelva a suspender el pellet en 1 ml de PBS 1X y el recuento de las células usando un hemocitómetro. </li><li> La transferencia de 1,5 x 10 6 células en un tubo centrífugo de 1,5 ml. Centrifugar a 300 xg durante 5 min. </li><li> Aspirar el sobrenadante con cuidado. Resuspender el pellet en 300 L de R Buffer (Sistema de neón de transfección, Invitrogen) y 6 mg (2 mg / 5,0 x 10 5 células) del P231-pCMVe betaAc-STOP-luc (Addgene, Cambridge, MA). Coloque 3 ml de tampón E (Invitrogen) en el puerto de atraque de la electroporación por el protocolo del fabricante (sistema de transfección de neón, Invitrogen). </li><li> La transferencia de células plásmido solución a un 100 l punta de pipeta de neón y electroporar con una duración de pulso de 20 ms y una magnitud de 1,500 voltios. Lugar electroporated células en un tubo cónico de 15 ml que contiene 9,7 ml α-MEM medio completo. </li><li> Repita el paso 1,5 y dos veces más células transfectadas piscina para producir un volumen total de 10 ml. Añadir 10 ml de la suspensión de células (1,5 x 10 6 células) a un matraz de 10 ml que contiene T175 α-MEM medio completo. La viabilidad celular después de la electroporación es de aproximadamente 30%, para producir aproximadamente 4,5 x 10 5 células viables por T175. </li><li> Cambiar α-MEM medio completo 24 horas después de la transfección. </li><li> La cosecha, cuando las células transfectadas 70 a 80% confluente (~ 2 - 3 días después de la electroporación). Realizar el recuento de células con un hemocitómetro y resuspender las células a una concentración de 1,0 x 10 6cells/50 L de α-MEM medio completo. Minimizar el tiempo que pasan las células en suspensión para reducir la muerte celular antes de la inyección. </li></ol> 2. Inyección intramiocárdica <ol><li> Inducir la anestesia por isoflurano (Phoenix Pharmaceuticals, Inc., St. Joseph, MO) en ratones transgénicos diseñados para expresar constitutivamente recombinasa Cre en todas las células (B6.C-Tg (CMV-cre) 1IGN / J, Jackson Laboratory, Bar Harbor, ME). </li><li> Eliminar el vello de la región torácica con cortar el pelo o la depilación química. </li><li> Intubar con un catéter de calibre 18 (Becton Dickinson &amp; Co, Franklin Lakes NJ) y el lugar en el ventilador del ratón en 120 a 130 respiraciones por minuto, con un volumen de movimiento de 150 mL. </li><li> Realice una incisión lateral en el cuarto espacio intercostal lo que produce una toracotomía. </li><li> Visualizar el corazón, hacer dos inyecciones de 25 l de suspensión de células transfectadas con una jeringa de 1 ml (Temuro Medical Corporation, Somerset, NJ) y aguja de calibre 28 (Becten Dickinson &amp; Co, Franklin Lakes NJ). Para facilitar la inyección intramiocárdica y evitar un daño excesivo al órgano, doblar la cabeza de la aguja a 90 grados. </li><li> Después de la inyección, uso de suturas absorbibles (por ejemplo, vicryl) para cerrar las costillas y las capas musculares. La piel de sutura cierra con nailon 4-0 o seda. </li><li> Permitir el ratón para recuperarse de la anestesia y la extubación. </li><li> Los grupos de control debe incluir los ratones que recibieron inyecciones Cre único medio, los ratones Cre recibir la misma concentración de células no transfectadas y ratones de tipo salvaje que reciben las células transfectadas (alternativamente, los ratones que recibieron células transfectadas Cre que no son propensas a fusionar). </li></ol> 3. Imagen biofotónica <ol><li> Cinco a quince minutos antes de exponer, por vía intraperitoneal (IP) inyectar 10 l por gramo de peso del cuerpo del ratón de 15 mg / ml de D-luciferina (Caliper Life Sciences, Hopkinton, MA). </li><li> Inducir la anestesia con isoflurano en ratones a través de un 4% para la inducción de unad 1 - 2% para el mantenimiento. </li><li> Coloque en posición supina en la caja de imágenes con máscara administrar un ratón - 2% para el mantenimiento de la anestesia con isoflurano (Sistema de Xenogen imágenes Biofotonico, Hopkinton, MA). Varios ratones se pueden visualizar al mismo tiempo. Imagen simulada de control del ratón con ratones de experimentación para facilitar la comparación de la señal luminiscente. </li><li> El uso de software de estar de la imagen (Xenogen), establecer el tiempo de exposición adecuado (normalmente 60 segundos, ver Resultados). Establece el área de imagen para adaptarse a los ratones y mantener el área constante a través de imágenes para evitar cambios en la sensibilidad. Fijar la altura de sujetos a 4,5 cm. </li><li> Adquirir la señal de luminiscencia de intensidad correspondiente a ratón o ratones en el campo de vista y guardar los archivos sin modificar la imagen. Imágenes del proceso para eliminar la señal de fondo que corresponde a un control o un ratón no manipulado. Valores de intensidad por encima del fondo corresponden a células fusionadas dentro del animal. Análisis de la intensidad puede ser llevado a cabo utilizando el software de vida de la imagen (Xenogen) o software de código abierto de análisis de imagen de origen. Typically, una región de interés (ROI) es seleccionado para comparar los datos de intensidad entre los animales y los experimentos. </li></ol> 4. Resultados representante Para determinar la sensibilidad del sistema de imágenes Biofotonico Xenogen, una línea celular que expresa constitutivamente la luciferasa (LUC-231-D3H1, Xenogen) fue entregado en el miocardio de C57/Bl6 ratones (Jackson Laboratory). Las células fueron inyectadas en concentraciones de 1 x 10 6, 1 x 10 3, o 1 x 10 1 en las células. Seis horas después del parto, células, los ratones fueron inyectados por vía intraperitoneal con la luciferina y la imagen utilizando el sistema de Xenogen. Una señal específica podría ser detectada con 1.000 células (2 de 6 ratones imágenes, la figura 2), pero la detección es más fiable con 10.000 células (6 de 6 ratones imágenes). Es importante destacar que este estudio también sirvió para establecer una correlación aproximada entre el número de células que expresan luciferasa y la intensidad de la señal. <p class="jove_content"&gt; Para demostrar la utilidad del protocolo descrito en la detección y el seguimiento de la fusión de células, las MSC se transfectaron con el plásmido LoxP-Stop-LoxP-luciferasa (Addgene) y entregados al miocardio de Cre-expresando ratones. Aproximadamente una semana después de la entrega de células, los ratones fueron estudiados primero utilizando el sistema de Xenogen sin D-luciferina de la inyección. Como era de esperar, sin el sustrato enzimático, no se detectó señal de intensidad (Figura 3). A continuación, D-luciferina se inyectó por vía intraperitoneal y una señal correspondiente a la intensidad de la luciferasa y por lo tanto la fusión celular fue detectado en dos de los cuatro ratones estudiados. Una señal similar se detectó una semana más tarde (Figura 3), lo que sugiere MSC-junto productos de fusión se pueden mantener en vivo. En este caso, el estudio se suspendió para permitir la evaluación del corazón y los tejidos circundantes, pero uno podría imaginar a largo plazo Los análisis y las imágenes más frecuentes para seguir el mantenimiento, la proliferación de unad tal vez la migración de productos de fusión en los ratones. <img alt="Figura 1" src="/files/ftp_upload/3581/3581fig1.jpg" /> Figura 1. Esquemática de la técnica para detectar la fusión de células in vivo. Si la fusión entre Cre-que expresan las células de ratón y células trasplantadas expresar un plásmido floxed luciferasa se ​​produce, la luciferasa se ​​expresa. Luciferasa se pueden detectar mediante la inyección del sustrato enzimático, D-luciferina, en el ratón y el ratón de imágenes usando un sistema de Xenogen imágenes Biofotonico (Adaptado de 19) <img alt="Figura 2" src="/files/ftp_upload/3581/3581fig2.jpg" /> Figura 2. Sensibilidad de la detección de la luciferasa que expresan las células en el tejido cardíaco con la imagen biofotónica. Una línea celular que expresa constitutivamente la luciferasa (LUC-231-D3H1, Xenogen) fue entregado al espacio intramiocárdica de C57/Bl6 ratones en varios números de celular total. Imágenes representativas de los ratones receiving 1 x 10 6, 1 x 10 3 y 1 x 10 1 en las células (de izquierda a derecha) se muestran, las imágenes se llevó a cabo aproximadamente 6 horas después de la inyección. <img alt="Figura 3" src="/files/ftp_upload/3581/3581fig3.jpg" /> Figura 3. La cuantificación de la luminiscencia en Vivo indicativo de la fusión celular. MSC fueron transfectadas con el LoxP-Stop-LoxP-Luciferase plásmido y se entregan en el miocardio de Cre-expresando ratones. Aproximadamente una semana y dos semanas después de la entrega de células, los ratones Cre fueron estudiados mediante el sistema de imagen Xenogen Biofotonico para medir la intensidad de la luminiscencia indicativo de la fusión celular. (A) Superposición de la fotografía y la intensidad de la luminiscencia de la farsa y ratones 1-4 (de izquierda a derecha) 17 días después de la entrega de la célula. (B) la intensidad de la luminiscencia de la farsa y ratones 4.1 (de izquierda a derecha) 17 días después de la entrega de la célula. Una región de interés ha sido seleccionada (amarillo), correspondiente a la zona de inyección y la intensidad de levels se determinaron utilizando ImageJ (gratuito) de software 20. (C) La intensidad de la luminiscencia se normalizó a la misma región de interés en la farsa del ratón para todas las condiciones experimentales. En una semana, los ratones 3 y 4 muestran la señal de luminiscencia positiva que sugiere la fusión espontánea de una célula del ratón y el MSC trasplantado. La señal persistía en el ratón 3 en dos semanas. Para determinar órgano-específicas de localización de la señal correspondiente al ratón de 3, de la cavidad torácica fue expuesto y de los órganos extirpados primaria y la imagen. (D) Superposición de la fotografía y la intensidad de la luminiscencia de las 3 del ratón. Tenga en cuenta la localización de la intensidad de la señal en el intestino delgado. (E) La intensidad de la luminiscencia de las 3 del ratón. (F) de superposición de la fotografía y la intensidad de la luminiscencia de la farsa del ratón. (G) La intensidad de la luminiscencia de la farsa del ratón.

<ol>
	<li>Bernirschke, K. K. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Pathology of the Human Placenta. , (2000).</li><li>Hoshina, M., Boothby, M., Boime, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cytological+localization+of+chorionic+gonadotropin+alpha+and+placental+lactogen+mRNAs+during+development+of+the+human+placenta.">Cytological localization of chorionic gonadotropin alpha and placental lactogen mRNAs during development of the human placenta.</a> J. Cell. Biol. 93, 190-198 (1982).</li><li>Johansen, M., Redman, C. W., Wilkins, T., Sargent, I. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Trophoblast+deportation+in+human+pregnancy--its+relevance+for+pre-eclampsia.">Trophoblast deportation in human pregnancy--its relevance for pre-eclampsia.</a> Placenta. 20, 531-539 (1999).</li><li>Redman, C. W., Sargent, I. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Placental+debris,+oxidative+stress+and+pre-eclampsia.">Placental debris, oxidative stress and pre-eclampsia.</a> Placenta. 21, 597-602 (2000).</li><li>Ogle, B. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spontaneous+fusion+of+cells+between+species+yields+transdifferentiation+and+retroviral+transfer+in+vivo.">Spontaneous fusion of cells between species yields transdifferentiation and retroviral transfer in vivo.</a> FASEB. J. 18, 548-550 (2004).</li><li>Nygren, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bone+marrow-derived+hematopoietic+cells+generate+cardiomyocytes+at+a+low+frequency+through+cell+fusion,+but+not+transdifferentiation.">Bone marrow-derived hematopoietic cells generate cardiomyocytes at a low frequency through cell fusion, but not transdifferentiation.</a> Nat. Med. 10, 494-501 (2004).</li><li>Nygren, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Myeloid+and+lymphoid+contribution+to+non-haematopoietic+lineages+through+irradiation-induced+heterotypic+cell+fusion.">Myeloid and lymphoid contribution to non-haematopoietic lineages through irradiation-induced heterotypic cell fusion.</a> Nat. Cell. Biol. 10, 584-592 (2008).</li><li>Kutschka, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adenoviral+human+BCL-2+transgene+expression+attenuates+early+donor+cell+death+after+cardiomyoblast+transplantation+into+ischemic+rat+hearts.">Adenoviral human BCL-2 transgene expression attenuates early donor cell death after cardiomyoblast transplantation into ischemic rat hearts.</a> Circulation. 114, I174-I180 (2006).</li><li>Min, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+bioluminescence+imaging+of+cord+blood+derived+mesenchymal+stem+cell+transplantation+into+rat+myocardium.">In vivo bioluminescence imaging of cord blood derived mesenchymal stem cell transplantation into rat myocardium.</a> Ann. Nucl. Med. 20, 165-170 (2006).</li><li>Malstrom, S. E., Tornavaca, O., Meseguer, A., Purchio, A. F., West, D. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+characterization+and+hormonal+regulation+of+kidney+androgen-regulated+protein+(Kap)-luciferase+transgenic+mice.">The characterization and hormonal regulation of kidney androgen-regulated protein (Kap)-luciferase transgenic mice.</a> Toxicol. Sci. 79, 266-277 (2004).</li><li>Weir, L. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biophotonic+imaging+in+HO-1.luc+transgenic+mice:+real-time+demonstration+of+gender-specific+chloroform+induced+renal+toxicity.">Biophotonic imaging in HO-1.luc transgenic mice: real-time demonstration of gender-specific chloroform induced renal toxicity.</a> Mutat. Res. 574, 67-75 (2005).</li><li>Rajashekara, G., Glover, D. A., Banai, M., O'Callaghan, D., Splitter, G. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Attenuated+bioluminescent+Brucella+melitensis+mutants+GR019+(virB4),+GR024+(galE),+and+GR026+(BMEI1090-BMEI1091)+confer+protection+in+mice.">Attenuated bioluminescent Brucella melitensis mutants GR019 (virB4), GR024 (galE), and GR026 (BMEI1090-BMEI1091) confer protection in mice.</a> Infect. Immun. 74, 2925-2936 (1090).</li><li>Kadurugamuwa, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Noninvasive+biophotonic+imaging+for+monitoring+of+catheter-associated+urinary+tract+infections+and+therapy+in+mice.">Noninvasive biophotonic imaging for monitoring of catheter-associated urinary tract infections and therapy in mice.</a> Infect. Immun. 73, 3878-3887 (2005).</li><li>Ryan, P. L., Youngblood, R. C., Harvill, J., Willard, S. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Photonic+monitoring+in+real+time+of+vascular+endothelial+growth+factor+receptor+2+gene+expression+under+relaxin-induced+conditions+in+a+novel+murine+wound+model.">Photonic monitoring in real time of vascular endothelial growth factor receptor 2 gene expression under relaxin-induced conditions in a novel murine wound model.</a> Ann. N.Y. Acad. Sci. 1041, 398-414 (2005).</li><li>Zhu, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Non-invasive+imaging+of+GFAP+expression+after+neuronal+damage+in+mice.">Non-invasive imaging of GFAP expression after neuronal damage in mice.</a> Neurosci. Lett. 367, 210-212 (2004).</li><li>Noiseux, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mesenchymal+stem+cells+overexpressing+Akt+dramatically+repair+infarcted+myocardium+and+improve+cardiac+function+despite+infrequent+cellular+fusion+or+differentiation.">Mesenchymal stem cells overexpressing Akt dramatically repair infarcted myocardium and improve cardiac function despite infrequent cellular fusion or differentiation.</a> Mol. Ther. 14, 840-850 (2006).</li><li>Ajiki, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Composite+tissue+transplantation+in+rats:+fusion+of+donor+muscle+to+the+recipient+site.">Composite tissue transplantation in rats: fusion of donor muscle to the recipient site.</a> Transplant Proc. 37, 208-209 (2005).</li><li>Trivedi, P., Hematti, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Derivation+and+immunological+characterization+of+mesenchymal+stromal+cells+from+human+embryonic+stem+cells.">Derivation and immunological characterization of mesenchymal stromal cells from human embryonic stem cells.</a> Exp. Hematol. 36, 350-359 (2008).</li><li>Ogle, B. M., Cascalho, M., Platt, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biological+implications+of+cell+fusion.">Biological implications of cell fusion.</a> Nat. Rev. Mol. Cell. Biol. 6, 567-575 (2005).</li><li>Collins, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ImageJ+for+microscopy.">ImageJ for microscopy.</a> BioTechniques. 43, 25-30 (2007).</li></ol>

No hay conflictos de interés declarado.

El método aquí descrito permite, por primera vez, la identificación y el análisis discreto temporal de la fusión de células en los organismos, incluyendo a los animales pequeños. El enfoque combina Cre-loxP recombinación con el posterior análisis de imagen biofotónica. El enfoque puede ser objeto de seguimiento no sólo la fusión entre células, sino también la fusión virus-célula y por lo tanto podría ser útil para el seguimiento de las infecciones virales. Análisis de imágenes es rápida y es posible...

<table border="1"><tbody><tr><td> Nombre del reactivo </td><td> Empresa </td><td> Número de catálogo </td><td> Comentarios </td></tr><tr><td> Neon sistema de transfección </td><td> Invitrogen, Carlsbad, CA </td><td> MPK5000 </td><td></td></tr><tr><td> Neon 100 Kit l </td><td> Invitrogen, Carlsbad, CA </td><td> MPK10025 </td><td> Contiene Buffer R y E </td></tr><tr><td> a-MEM en polvo </td><td> Invitrogen, Carlsbad, CA </td><td> 12000-022 </td><td></td></tr><tr><td> De suero fetal bovino (FBS) </td><td> Hyclone, Logan UT </td><td> SH30070.03 </td><td></td></tr><tr><td> B6.C-Tg (CMV-cre) 1IGN / J </td><td> Jackson Laboratory, Bar Harbor, ME </td><td> 006054 </td><td></td></tr><tr><td> 10 veces la tripsina </td><td> Fisher Scientific, Forest Lawn, NJ </td&gt; <td> MT-25-054-Cl </td><td></td></tr><tr><td> L-glutamina </td><td> Fisher Scientific, Forest Lawn, NJ </td><td> 25030-081 </td><td></td></tr><tr><td> D-luciferina </td><td> Caliper Life Sciences, Hopkinton, MA </td><td> 122796 </td><td></td></tr><tr><td> Xenogen Imaging System Biofotonico </td><td> Caliper Life Sciences, Hopkinton, MA </td><td> IVIS del espectro </td><td></td></tr><tr><td> Sodio Biocarbonate </td><td> Sigma Aldrich, St. Louis, MO </td><td> S6014-500G </td><td></td></tr><tr><td> No esenciales Aminoácidos </td><td> Invitrogen, Carlsbad, CA </td><td> 11140-050 </td><td></td></tr></tbody></table>

a cre-lox p recombination approach for the detection of cell fusion in vivo

La capacidad de dos o más células del mismo tipo de fusible se ha utilizado en los metazoos largo de la evolución para formar muchos órganos complejos, incluyendo el músculo esquelético, hueso y placenta. Los estudios contemporáneos demuestran la fusión de células del mismo tipo que le confiere la función mejorada. Por ejemplo, cuando las células trofoblásticas de la placenta el fusible para formar el sincitiotrofoblasto, el sincitiotrofoblasto está en mejores condiciones para el transporte de nutrientes y hormonas a través de la barrera materno-fetal del trofoblasto sin fundir 1-4. Estudios más recientes demuestran que la fusión de células de diferentes tipos pueden dirigir el destino celular. El &quot;retorno&quot; o la modificación de destino de la célula por fusión alguna vez se pensó que limitarse a los sistemas de cultivo celular. Pero el advenimiento del trasplante de células madre llevó al descubrimiento por nosotros y otros que las células madre pueden fusionarse con las células somáticas in vivo y que la fusión facilita la diferenciación de células madre 7.5. Por lo tanto, la fusión celular es un proceso regulado capable de promover la supervivencia y la diferenciación celular y por lo tanto podría ser de vital importancia para el desarrollo, la reparación de los tejidos e incluso la patogénesis de la enfermedad. La limitación del estudio de la fusión de células, es la falta de tecnología adecuada para 1) identificar con precisión los productos de la fusión y 2) los productos de la pista de fusión con el tiempo. Aquí se presenta un nuevo enfoque para abordar tanto las limitaciones a través de la inducción de la bioluminiscencia en la fusión (Figura 1);. Bioluminiscencia se puede detectar con alta sensibilidad en vivo 15.08 Utilizamos una construcción que codifica para la luciferasa de luciérnaga (Photinus pyralis) de genes adyacentes a un codón de parada, flanqueada por secuencias loxP. Cuando las células que expresan este gen fusionan con las células que expresan la proteína recombinasa Cre, los sitios loxP se rompen y la señal de parada se extirpa lo que permite la transcripción de la luciferasa. Debido a que la señal es inducible, la incidencia de falsos positivos señales es muy baja. A diferencia de los métodos existentes que utilizan el sistema Cre / LoxP 16, 17, hemos incorporado un &quot;vivo&quot; de detección de señales y por lo tanto pagar por primera vez la oportunidad para hacer un seguimiento de la cinética de la fusión de células in vivo. Para demostrar el enfoque, los ratones doquier expresar la recombinasa Cre se desempeñó como receptores de las células madre transfectadas con una construcción de expresar luciferasa aguas abajo de un codón de parada floxed. Las células madre fueron trasplantadas a través de la inyección intramiocárdica y después del trasplante de análisis de imagen intravital se llevó a cabo para rastrear la presencia de productos de fusión en el corazón y los tejidos circundantes a través del tiempo. Este enfoque podría adaptarse para analizar la fusión de células en cualquier tipo de tejido en cualquier etapa del desarrollo, la enfermedad o la reparación de tejidos adultos.

Watch this Scientific Journal Video about A Cre-Lox P Recombination Approach for the Detection of Cell Fusion In Vivo at JoVE.com

A Cre-Lox P Recombination Approach for the Detection of Cell Fusion In Vivo

Un método para hacer un seguimiento de la fusión celular en los organismos vivos con el tiempo se describe. El enfoque utiliza Cre-<em&gt; LoxP</emRecombinación&gt; para inducir la expresión de luciferasa en la fusión de células. La señal luminosa generada puede ser detectado en los organismos vivos mediante sistemas de biofotónica imágenes con una sensibilidad de detección de ~ 1.000 células en los tejidos periféricos.

A Cre-Lox P Enfoque de recombinación para la detección de la fusión celular En Vivo</em

The ability of two or more cells of the same type to fuse has been utilized in metazoans throughout evolution to form many complex organs, including ...

a-cre-lox-p-recombination-approach-for-detection-cell-fusion

Research

JoVE Journal

Bioengineering

A Cre-Lox P Recombination Approach for the Detection of Cell Fusion In Vivo

18.7K vistas.  University of Wisconsin-Madison. Un método para hacer un seguimiento de la fusión celular en los organismos vivos con el tiempo se describe. El enfoque utiliza Cre- LoxP para inducir la expresión de luciferasa en la fusión de células. La señal luminosa generada puede ser detectado en los organismos vivos mediante sistemas de biofotónica imágenes con una sensibilidad de detección de ~ 1.000 células en los tejidos periféricos.

Video: A Cre-Lox P Recombination Approach for the Detection of Cell Fusion In Vivo

Biomolecular Detection employing the Interferometric Reflectance Imaging Sensor (IRIS)

The sensitive measurement of biomolecular interactions has use in many fields and industries such as basic biology and microbiology, environmental/agricultural/biodefense monitoring, nanobiotechnology, and more. For diagnostic applications, monitoring (detecting) the presence, absence, or abnormal expression of targeted proteomic or genomic biomarkers found in patient samples can be used to determine treatment approaches or therapy efficacy. In the research arena, information on molecular affinities and specificities are useful for fully characterizing the systems under investigation.
Many of the current systems employed to determine molecular concentrations or affinities rely on the use of labels. Examples of these systems include immunoassays such as the enzyme-linked immunosorbent assay (ELISA), polymerase chain reaction (PCR) techniques, gel electrophoresis assays, and mass spectrometry (MS). Generally, these labels are fluorescent, radiological, or colorimetric in nature and are directly or indirectly attached to the molecular target of interest. Though the use of labels is widely accepted and has some benefits, there are drawbacks which are stimulating the development of new label-free methods for measuring these interactions. These drawbacks include practical facets such as increased assay cost, reagent lifespan and usability, storage and safety concerns, wasted time and effort in labelling, and variability among the different reagents due to the labelling processes or labels themselves. On a scientific research basis, the use of these labels can also introduce difficulties such as concerns with effects on protein functionality/structure due to the presence of the attached labels and the inability to directly measure the interactions in real time.
Presented here is the use of a new label-free optical biosensor that is amenable to microarray studies, termed the Interferometric Reflectance Imaging Sensor (IRIS), for detecting proteins, DNA, antigenic material, whole pathogens (virions) and other biological material. The IRIS system has been demonstrated to have high sensitivity, precision, and reproducibility for different biomolecular interactions [1-3]. Benefits include multiplex imaging capacity, real time and endpoint measurement capabilities, and other high-throughput attributes such as reduced reagent consumption and a reduction in assay times. Additionally, the IRIS platform is simple to use, requires inexpensive equipment, and utilizes silicon-based solid phase assay components making it compatible with many contemporary surface chemistry approaches.
Here, we present the use of the IRIS system from preparation of probe arrays to incubation and measurement of target binding to analysis of the results in an endpoint format. The model system will be the capture of target antibodies which are specific for human serum albumin (HSA) on HSA-spotted substrates.

Biomolecular Detection employing the Interferometric Reflectance Imaging Sensor IRIS

Quantitative, high-throughput, real-time, and label-free biomolecular detection (DNA, protein, etc.) on SiO2 surfaces can be achieved using a simple interferometric technique which relies on LED illumination, minimal optical components, and a camera. The Interferometric Reflectance Imaging Sensor (IRIS) is inexpensive, simple to use, and amenable to microarray formats.

Biomoleculares de detección utilizando el sensor de reflectancia de imágenes interferométricas (IRIS)

The sensitive measurement of biomolecular interactions has use in many fields and industries such as basic biology and microbiology, ...

Investigación

Bioingeniería

Fabrication of Electrochemical-DNA Biosensors for the Reagentless Detection of Nucleic Acids, Proteins and Small Molecules

As medicine is currently practiced, doctors send specimens to a central laboratory for testing and thus must wait hours or days to 
receive the results. Many patients would be better served by rapid, bedside tests. To this end our laboratory and others have developed a versatile, reagentless 
biosensor platform that supports the quantitative, reagentless, electrochemical detection of nucleic acids (DNA, RNA), proteins (including antibodies) and small 
molecules analytes directly in unprocessed clinical and environmental samples. In this video, we demonstrate the preparation and use of several biosensors in this 
"E-DNA" class. In particular, we fabricate and demonstrate sensors for the detection of a target DNA sequence in a polymerase chain reaction mixture, an HIV-specific antibody and the drug cocaine. The preparation procedure requires only three hours of hands-on effort followed by an overnight incubation, and their use requires only minutes.

"E-DNA" sensors, reagentless, electrochemical biosensors that perform well even when challenged directly in blood and other complex matrices, have been adapted to the detection of a wide range of nucleic acid, protein and small molecule analytes. Here we present a general procedure for the fabrication and use of such sensors.

Fabricación de ADN electroquímico biosensores para la detección Reagentless de ácidos nucleicos, proteínas y moléculas pequeñas

As medicine is currently practiced, doctors send specimens to a central laboratory for testing and thus must wait hours or days to 
receive the results. ...

Creating Two-Dimensional Patterned Substrates for Protein and Cell Confinement

Microcontact printing provides a rapid, highly reproducible method for the creation of well-defined patterned substrates.1 While microcontact printing can be employed to directly print a large number of molecules, including proteins,2 DNA,3 and silanes,4 the formation of self-assembled monolayers (SAMs) from long chain alkane thiols on gold provides a simple way to confine proteins and cells to specific patterns containing adhesive and resistant regions. This confinement can be used to control cell morphology and is useful for examining a variety of questions in protein and cell biology. Here, we describe a general method for the creation of well-defined protein patterns for cellular studies.5 This process involves three steps: the production of a patterned master using photolithography, the creation of a PDMS stamp, and microcontact printing of a gold-coated substrate. Once patterned, these cell culture substrates are capable of confining proteins and/or cells (primary cells or cell lines) to the pattern.
The use of self-assembled monolayer chemistry allows for precise control over the patterned protein/cell adhesive regions and non-adhesive regions; this cannot be achieved using direct protein stamping. Hexadecanethiol, the long chain alkane thiol used in the microcontact printing step, produces a hydrophobic surface that readily adsorbs protein from solution. The glycol-terminated thiol, used for backfilling the non-printed regions of the substrate, creates a monolayer that is resistant to protein adsorption and therefore cell growth.6 These thiol monomers produce highly structured monolayers that precisely define regions of the substrate that can support protein adsorption and cell growth. As a result, these substrates are useful for a wide variety of applications from the study of intercellular behavior7 to the creation of microelectronics.8
While other types of monolayer chemistry have been used for cell culture studies, including work from our group using trichlorosilanes to create patterns directly on glass substrates,9 patterned monolayers formed from alkane thiols on gold are straight-forward to prepare. Moreover, the monomers used for monolayer preparation are commercially available, stable, and do not require storage or handling under inert atmosphere. Patterned substrates prepared from alkane thiols can also be recycled and reused several times, maintaining cell confinement.10

Self-assembled monolayers (SAMs) formed from long chain alkane thiols on gold provide well-defined substrates for the formation of protein patterns and cell confinement. Microcontact printing of hexadecanethiol using a polydimethylsiloxane (PDMS) stamp followed by backfilling with a glycol-terminated alkane thiol monomer produces a pattern where protein and cells adsorb only to the stamped hexadecanethiol region.

La creación de dos dimensiones con dibujos sustratos para el confinamiento de proteínas y células

Microcontact printing provides a rapid, highly reproducible method for the creation of well-defined patterned substrates.1  While microcontact printing ...

Simple Microfluidic Devices for in vivo Imaging of C. elegans, Drosophila and Zebrafish

Micro fabricated fluidic devices provide an accessible micro-environment for in vivo studies on small organisms. Simple fabrication processes are available for microfluidic devices using soft lithography techniques 1-3. Microfluidic devices have been used for sub-cellular imaging 4,5, in vivo laser microsurgery 2,6 and cellular imaging 4,7. In vivo imaging requires immobilization of organisms. This has been achieved using suction 5,8, tapered channels 6,7,9, deformable membranes 2-4,10, suction with additional cooling 5, anesthetic gas 11, temperature sensitive gels 12, cyanoacrylate glue 13 and anesthetics such as levamisole 14,15. Commonly used anesthetics influence synaptic transmission 16,17 and are known to have detrimental effects on sub-cellular neuronal transport 4. In this study we demonstrate a membrane based poly-dimethyl-siloxane (PDMS) device that allows anesthetic free immobilization of intact genetic model organisms such as Caenorhabditis elegans (C. elegans), Drosophila larvae and zebrafish larvae. These model organisms are suitable for in vivo studies in microfluidic devices because of their small diameters and optically transparent or translucent bodies. Body diameters range from ~10 &mu;m to ~800 &mu;m for early larval stages of C. elegans and zebrafish larvae and require microfluidic devices of different sizes to achieve complete immobilization for high resolution time-lapse imaging. These organisms are immobilized using pressure applied by compressed nitrogen gas through a liquid column and imaged using an inverted microscope. Animals released from the trap return to normal locomotion within 10 min.
We demonstrate four applications of time-lapse imaging in C. elegans namely, imaging mitochondrial transport in neurons, pre-synaptic vesicle transport in a transport-defective mutant, glutamate receptor transport and Q neuroblast cell division. Data obtained from such movies show that microfluidic immobilization is a useful and accurate means of acquiring in vivo data of cellular and sub-cellular events when compared to anesthetized animals (Figure 1J and 3C-F 4).
Device dimensions were altered to allow time-lapse imaging of different stages of C. elegans, first instar Drosophila larvae and zebrafish larvae. Transport of vesicles marked with synaptotagmin tagged with GFP (syt.eGFP) in sensory neurons shows directed motion of synaptic vesicle markers expressed in cholinergic sensory neurons in intact first instar Drosophila larvae. A similar device has been used to carry out time-lapse imaging of heartbeat in ~30 hr post fertilization (hpf) zebrafish larvae. These data show that the simple devices we have developed can be applied to a variety of model systems to study several cell biological and developmental phenomena in vivo.

Simple Microfluidic Devices for in vivo Imaging of C. elegans, Drosophila and Zebrafish

A simple microfluidic device has been developed to perform anesthetic free in vivo imaging of C. elegans, intact Drosophila larvae and zebrafish larvae. The device utilizes a deformable PDMS membrane to immobilize these model organisms in order to perform time lapse imaging of numerous processes such as heart beat, cell division and sub-cellular neuronal transport. We demonstrate the use of this device and show examples of different types of data collected from different model systems.

Simples dispositivos de microfluidos para<em&gt; In vivo</em&gt; Imágenes de<em&gt; C. elegans</em&gt;,<em&gt; Drosophila</em&gt; Y pez cebra

Micro fabricated fluidic devices provide an accessible micro-environment for in vivo studies on small organisms. Simple fabrication processes are ...

Microinjection Wound Assay and In vivo Localization of Epidermal Wound Response Reporters in Drosophila Embryos.

The Drosophila embryo develops a robust epidermal layer that serves both to protect the internal cells from a harsh external environment as well as to maintain cellular homeostasis. Puncture injury with glass needles provides a direct method to trigger a rapid epidermal wound response that activates wound transcriptional reporters, which can be visualized by a localized reporter signal in living embryos or larvae. Puncture or laser injury also provides signals that promote the recruitment of hemocytes to the wound site. Surprisingly, severe (through and through) puncture injury in late stage embryos only rarely disrupts normal embryonic development, as greater than 90% of such wounded embryos survive to adulthood when embryos are injected in an oil medium that minimizes immediate leakage of hemolymph from puncture sites. The wound procedure does require micromanipulation of the Drosophila embryos, including manual alignment of the embryos on agar plates and transfer of the aligned embryos to microscope slides. The Drosophila epidermal wound response assay provides a quick system to test the genetic requirements of a variety of biological functions that promote wound healing, as well as a way to screen for potential chemical compounds that promote wound healing. The short life cycle and easy culturing routine make Drosophila a powerful model organism. Drosophila clean wound healing appears to coordinate the epidermal regenerative response, with the innate immune response, in ways that are still under investigation, which provides an excellent system to find conserved regulatory mechanisms common to Drosophila and mammalian epidermal wounding.

Microinjection Wound Assay and In vivo Localization of Epidermal Wound Response Reporters in Drosophila Embryos.

The embryonic epidermis of very late stage Drosophila embryos provides an in vivo system for rapid puncture wound response analysis and can be combined with genetic manipulations or chemical microinjection treatments to advance studies in wound healing for translation into mammalian models.

Microinyección de Heridas Ensayo y<em&gt; In vivo</em&gt; Localización de la herida epidérmica Reporteros de respuesta en<em&gt; Drosophila</em&gt; Embriones.

The Drosophila embryo develops a robust epidermal layer that serves both to protect the internal cells from a harsh external environment as well as to ...

A Full Skin Defect Model to Evaluate Vascularization of Biomaterials In Vivo

Insufficient vascularization is considered to be one of the main factors limiting the clinical success of tissue-engineered constructs. In order to evaluate new strategies that aim at improving vascularization, reliable methods are required to make the in-growth of new blood vessels into bio-artificial scaffolds visible and quantify the results. Over the past couple of years, our group has introduced a full skin defect model that enables the direct visualization of blood vessels by transillumination and provides the possibility of quantification through digital segmentation. In this model, one surgically creates full skin defects in the back of mice and replaces them with the material tested. Molecules or cells of interest can also be incorporated in such materials to study their potential effect. After an observation time of one&#8217;s own choice, materials are explanted for evaluation. Bilateral wounds provide the possibility of making internal comparisons that minimize artifacts among individuals as well as of decreasing the number of animals needed for the study. In comparison to other approaches, our method offers a simple, reliable and cost effective analysis. We have implemented this model as a routine tool to perform high-resolution screening when testing vascularization of different biomaterials and bio-activation approaches.

A Full Skin Defect Model to Evaluate Vascularization of Biomaterials In Vivo

Vascularization is key to approaches in successful tissue engineering. Therefore, reliable technologies are required to evaluate the development of vascular networks in tissue-constructs. Here we present a simple and cost-effective method to visualize and quantify vascularization in vivo.

Una piel llena de defectos modelo para evaluar la vascularización de Biomateriales<em&gt; En Vivo</em

Insufficient vascularization is considered to be one of the main factors limiting the clinical success of tissue-engineered constructs. In order to ...

A Label-free Technique for the Spatio-temporal Imaging of Single Cell Secretions

Inter-cellular communication is an integral part of a complex system that helps in maintaining basic cellular activities. As a result, the malfunctioning of such signaling can lead to many disorders. To understand cell-to-cell signaling, it is essential to study the spatial and temporal nature of the secreted molecules from the cell without disturbing the local environment. Various assays have been developed to study protein secretion, however, these methods are typically based on fluorescent probes which disrupt the relevant signaling pathways. To overcome this limitation, a label-free technique is required.
In this paper, we describe the fabrication and application of a label-free localized surface plasmon resonance imaging (LSPRi) technology capable of detecting protein secretions from a single cell. The plasmonic nanostructures are lithographically patterned onto a standard glass coverslip and can be excited using visible light on commercially available light microscopes. Only a small fraction of the coverslip is covered by the nanostructures and hence this technique is well suited for combining common techniques such as fluorescence and bright-field imaging.
A multidisciplinary approach is used in this protocol which incorporates sensor nanofabrication and subsequent biofunctionalization, binding kinetics characterization of ligand and analyte, the integration of the chip and live cells, and the analysis of the measured signal. As a whole, this technology enables a general label-free approach towards mapping cellular secretions and correlating them with the responses of nearby cells.

Inter-cellular communication is critical for controlling various physiological activities within and outside the cell. This paper describes a protocol for measuring the spatio-temporal nature of single cell secretions. To achieve this, a multidisciplinary approach is used which integrates label-free nanoplasmonic sensing with live cell imaging.

Una técnica-Label libre para el Imaging espacio-temporal de las secreciones sola célula

Inter-cellular communication is an integral part of a complex system that helps in maintaining basic cellular activities. As a result, the malfunctioning ...

Hollow Fiber Bioreactors for In Vivo-like Mammalian Tissue Culture

Tissue culture has been used for over 100 years to study cells and responses ex vivo. The convention of this technique is the growth of anchorage dependent cells on the 2-dimensional surface of tissue culture plastic. More recently, there is a growing body of data demonstrating more in vivo-like behaviors of cells grown in 3-dimensional culture systems. This manuscript describes in detail the set-up and operation of a hollow fiber bioreactor system for the in vivo-like culture of mammalian cells. The hollow fiber bioreactor system delivers media to the cells in a manner akin to the delivery of blood through the capillary networks in vivo. The system is designed to fit onto the shelf of a standard CO2 incubator and is simple enough to be set-up by any competent cell biologist with a good understanding of aseptic technique. The systems utility is demonstrated by culturing the hepatocarcinoma cell line HepG2/C3A for 7 days. Further to this and in line with other published reports on the functionality of cells grown in 3-dimensional culture systems the cells are shown to possess increased albumin production (an important hepatic function) when compared to standard 2-dimensional tissue culture.

Hollow Fiber Bioreactors for In Vivo-like Mammalian Tissue Culture

The functional behavior of cells in culture can be improved by culturing in more in vivo-like 3-dimensional culture environments16-21. This manuscript describes the set-up and operation of a hollow fiber bioreactor system for in vivo-like mammalian tissue culture.

Biorreactores de fibra hueca para<em&gt; En Vivo</em&gt; -como Cultivo de tejidos de mamíferos

Tissue culture has been used for over 100 years to study cells and responses ex vivo. The convention of this technique is the growth of anchorage ...

Preparation of 3D Collagen Gels and Microchannels for the Study of 3D Interactions In Vivo

Historically, most cellular processes have been studied in only 2 dimensions. While these studies have been informative about general cell signaling mechanisms, they neglect important cellular cues received from the structural and mechanical properties of the local microenvironment and extracellular matrix (ECM). To understand how cells interact within a physiological ECM, it is important to study them in the context of 3 dimensional assays. Cell migration, cell differentiation, and cell proliferation are only a few processes that have been shown to be impacted by local changes in the mechanical properties of a 3-dimensional ECM. Collagen I, a core fibrillar component of the ECM, is more than a simple structural element of a tissue. Under normal conditions, mechanical cues from the collagen network direct morphogenesis and maintain cellular structures. In diseased microenvironments, such as the tumor microenvironment, the collagen network is often dramatically remodeled, demonstrating altered composition, enhanced deposition and altered fiber organization. In breast cancer, the degree of fiber alignment is important, as an increase in aligned fibers perpendicular to the tumor boundary has been correlated to poorer patient prognosis1. Aligned collagen matrices result in increased dissemination of tumor cells via persistent migration2,3. The following is a simple protocol for embedding cells within a 3-dimensional, fibrillar collagen hydrogel. This protocol is readily adaptable to many platforms, and can reproducibly generate both aligned and random collagen matrices for investigation of cell migration, cell division, and other cellular processes in a tunable, 3-dimensional, physiological microenvironment.

Preparation of 3D Collagen Gels and Microchannels for the Study of 3D Interactions In Vivo

Collagen is a core component of the ECM, and provides essential cues for several cellular processes ranging from migration to differentiation and proliferation. Provided here is a protocol for embedding cells within 3D collagen hydrogels, and a more advanced technique for generating randomized or aligned collagen matrices using PDMS microchannels.

Preparación de geles de colágeno en 3D y microcanales para el estudio de las interacciones 3D<em&gt; En Vivo</em

Historically, most cellular processes have been studied in only 2 dimensions.  While these studies have been informative about general cell signaling ...

Visual Detection of Multiple Nucleic Acids in a Capillary Array

Multi-target, short time, and resource-affordable methodologies for the detection of multiple nucleic acids in a single, easy to operate test are urgently needed in disease diagnosis, microbial monitoring, genetically modified organism (GMO) detection, and forensic analysis. We have previously described the platform called CALM (Capillary Array-based Loop-mediated isothermal amplification for Multiplex visual detection of nucleic acids). Herein, we describe improved fabrication and performance processes for this platform. Here, we apply a small, ready-to-use cassette assembled by capillary array for multiplex visual detection of nucleic acids. The capillary array is pre-treated into a hydrophobic and hydrophilic pattern before fixing loop-mediated isothermal amplification (LAMP) primer sets in capillaries. After assembly of the loading adaptor, LAMP reaction mixture is loaded and isolated into each capillary, due to capillary force by a single pipetting step. The LAMP reactions are performed in parallel in the capillaries. The results are visually read out by illumination with a hand-held UV flashlight. Using this platform, we demonstrate monitoring of 8 frequently appearing elements and genes in GMO samples with high specificity and sensitivity. In summary, the platform described herein is intended to facilitate the detection of multiple nucleic acids. We believe it will be widely applicable in fields where high-throughput nucleic acid analysis is required.

This protocol describes the fabrication of a small, ready-to-use cassette that can be applied for visual detection of multiple nucleic acids in a single, test that is easy to operate. In this approach, a capillary array was used for multiplex and highly efficient detection of GMO targets.

Detección visual de los ácidos nucleicos múltiples en un arreglo de capilares

Multi-target, short time, and resource-affordable methodologies for the detection of multiple nucleic acids in a single, easy to operate test are urgently ...