Trabajo en el laboratorio los autores ha sido apoyado por el USDA / CSREES / NRICGP (2007-35203-18213), NIH / CNRR (2 R01 RR17359-06), NIH / NIEHS (1 R21 ES014856-01A2) y Alberta Innova - Soluciones para la Salud.

<ol>
	<li>Brinster, R. L., Avarbock, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Germline+transmission+of+donor+haplotype+following+spermatogonial+transplantation.">Germline transmission of donor haplotype following spermatogonial transplantation.</a> Proceedings of the National Academy of Sciences of the United States of America. 91 (24), 11303-11303 (1994).</li><li>Brinster, R. L., Zimmermann, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spermatogenesis+following+male+germ-cell+transplantation.">Spermatogenesis following male germ-cell transplantation.</a> Proceedings of the National Academy of Sciences of the United States of America. 91 (24), 11298-11298 (1994).</li><li>Nagano, M., Avarbock, M. R., Brinster, R. 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M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sertoli+cells+dictate+spermatogonial+stem+cell+niches+in+the+mouse+testis.">Sertoli cells dictate spermatogonial stem cell niches in the mouse testis.</a> Biology of Reproduction. 84 (4), 639-639 (2011).</li><li>Nagano, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Homing+efficiency+and+proliferation+kinetics+of+male+germ+line+stem+cells+following+transplantation+in+mice.">Homing efficiency and proliferation kinetics of male germ line stem cells following transplantation in mice.</a> Biology of Reproduction. 69 (2), 701-701 (2003).</li><li>Kubota, H., Avarbock, M. R., Brinster, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Growth+factors+essential+for+self-renewal+and+expansion+of+mouse+spermatogonial+stem+cells.">Growth factors essential for self-renewal and expansion of mouse spermatogonial stem cells.</a> Proceedings of the National Academy of Sciences of the United States of America. 101 (47), 16489-16489 (2004).</li><li>Oatley, J. M., Avarbock, M. R., Telaranta, A. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identifying+genes+important+for+spermatogonial+stem+cell+self-renewal+and+survival.">Identifying genes important for spermatogonial stem cell self-renewal and survival.</a> Proceedings of the National Academy of Sciences of the United States of America. 103 (25), 9524-9524 (2006).</li><li>Dobrinski, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Germ+cell+transplantation+and+testis+tissue+xenografting+in+domestic+animals.">Germ cell transplantation and testis tissue xenografting in domestic animals.</a> Animal Reproduction Science. 89 (1-4), 137-137 (2005).</li><li>Dobrinski, I., Avarbock, M. R., Brinster, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transplantation+of+germ+cells+from+rabbits+and+dogs+into+mouse+testes.">Transplantation of germ cells from rabbits and dogs into mouse testes.</a> Biology of Reproduction. 61 (5), 1331-1331 (1999).</li><li>Honaramooz, A., Snedaker, A., Boiani, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sperm+from+neonatal+mammalian+testes+grafted+in+mice.">Sperm from neonatal mammalian testes grafted in mice.</a> Nature. 418 (6899), 778-778 (2002).</li><li>Rodriguez-Sosa, J. R., Dobrinski, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recent+developments+in+testis+tissue+xenografting.">Recent developments in testis tissue xenografting.</a> Reproduction. 138 (2), 187-187 (2009).</li><li>Dobrinski, I., Rathi, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ectopic+grafting+of+mammalian+testis+tissue+into+mouse+hosts.">Ectopic grafting of mammalian testis tissue into mouse hosts.</a> Methods in Molecular Biology. 139, 450-450 (2008).</li><li>Rodriguez-Sosa, J. R., Schlatt, S., Dobrinski, I., Seli, E., Agarwal, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Testicular+tissue+transplantation+for+fertility+preservation.">Testicular tissue transplantation for fertility preservation.</a> Fertility Preservation: Emerging Technologies and Clinical Applications. , 331-331 (2011).</li><li>Rathi, R., Honaramooz, A., Zeng, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Germ+cell+fate+and+seminiferous+tubule+development+in+bovine+testis+xenografts.">Germ cell fate and seminiferous tubule development in bovine testis xenografts.</a> Reproduction. 130 (6), 923-923 (2005).</li><li>Rathi, R., Honaramooz, A., Zeng, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Germ+cell+development+in+equine+testis+tissue+xenografted+into+mice.">Germ cell development in equine testis tissue xenografted into mice.</a> Reproduction. 131 (6), 1091-1091 (2006).</li><li>Kim, Y., Selvaraj, V., Pukazhenthi, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+donor+age+on+success+of+spermatogenesis+in+feline+testis+xenografts.">Effect of donor age on success of spermatogenesis in feline testis xenografts.</a> Reproduction, Fertility, and Development. 19 (7), 869-869 (2007).</li><li>Arregui, L., Rathi, R., Zeng, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Xenografting+of+adult+mammalian+testis+tissue.">Xenografting of adult mammalian testis tissue.</a> Animal Reproduction Science. 106 (1-2), 65-65 (2008).</li><li>Schlatt, S., Honaramooz, A., Ehmcke, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Limited+survival+of+adult+human+testicular+tissue+as+ectopic+xenograft.">Limited survival of adult human testicular tissue as ectopic xenograft.</a> Human Reproduction. 21 (2), 384-384 (2006).</li><li>Shinohara, T., Avarbock, M. R., Brinster, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=beta1-+and+alpha6-integrin+are+surface+markers+on+mouse+spermatogonial+stem+cells.">beta1- and alpha6-integrin are surface markers on mouse spermatogonial stem cells.</a> Proceedings of the National Academy of Sciences of the United States of America. 96 (10), 5504-5504 (1999).</li><li>Kanatsu-Shinohara, M., Toyokuni, S., Shinohara, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CD9+is+a+surface+marker+on+mouse+and+rat+male+germline+stem+cells.">CD9 is a surface marker on mouse and rat male germline stem cells.</a> Biology of Reproduction. 70 (1), 70-70 (2004).</li><li>Ryu, B. Y., Orwig, K. E., Oatley, 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=Effects+of+aging+and+niche+microenvironment+on+spermatogonial+stem+cell+self-renewal.">Effects of aging and niche microenvironment on spermatogonial stem cell self-renewal.</a> Stem Cells. 24 (6), 1505-1505 (2006).</li><li>Costoya, J. A., Hobbs, R. M., Barna, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Essential+role+of+Plzf+in+maintenance+of+spermatogonial+stem+cells.">Essential role of Plzf in maintenance of spermatogonial stem cells.</a> Nature Genetics. 36 (6), 653-653 (2004).</li><li>Morrow, C. M., Hostetler, C. E., Griswold, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ETV5+is+required+for+continuous+spermatogenesis+in+adult+mice+and+may+mediate+blood+testes+barrier+function+and+testicular+immune+privilege.">ETV5 is required for continuous spermatogenesis in adult mice and may mediate blood testes barrier function and testicular immune privilege.</a> Annals of the New York Academy of Sciences. 1120, 144-144 (2007).</li><li>Zeng, W., Snedaker, A. K., Megee, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preservation+and+transplantation+of+porcine+testis+tissue.">Preservation and transplantation of porcine testis tissue.</a> Reproduction, Fertility and Development. 21 (3), 489-489 (2009).</li><li>Jahnukainen, K., Ehmcke, J., Hergenrother, S. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+cold+storage+and+cryopreservation+of+immature+non-human+primate+testicular+tissue+on+spermatogonial+stem+cell+potential+in+xenografts.">Effect of cold storage and cryopreservation of immature non-human primate testicular tissue on spermatogonial stem cell potential in xenografts.</a> Human Reproduction. 22 (4), 1060-1060 (2007).</li><li>Rathi, R., Zeng, W., Megee, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Maturation+of+testicular+tissue+from+infant+monkeys+after+xenografting+into+mice.">Maturation of testicular tissue from infant monkeys after xenografting into mice.</a> Endocrinology. 149 (10), 5288-5288 (2008).</li><li>Honaramooz, A., Li, M. W., Penedo, M. 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No hay conflictos de interés declarado.

1. El transplante de células germinales Germen de trasplante de células proporciona el ensayo sólo funcional para la confirmación inequívoca de la presencia de células madre de espermatogonias (SSC) en una población celular. SSC Sólo en casa pueden y colonizar el nicho de cooperación Sur-Sur en la membrana basal e iniciar los donantes derivado de la espermatogénesis. El transplante de células germinales ha permitido estudiar y manipular las ESC de una manera sin precedentes. La téc...

<table border="1"><tbody><tr><td> Nombre del reactivo </td><td> Empresa </td><td> Número de catálogo </td></tr><tr><td> La colagenasa (tipo IV) </td><td> Sigma </td><td> C5138 </td></tr><tr><td> Tripsina-EDTA </td><td> Invitrogen </td><td> 25200-056 </td></tr><tr><td> DNaseI </td><td> Sigma </td><td> DN25 </td></tr><tr><td> DMEM </td><td> Invitrogen </td><td> 31053-028 </td></tr><tr><td> Trypan mancha azul </td><td> Invitrogen </td><td> 15250-061 </td></tr><tr><td> Malla de nylon filtro de células </td><td> BD Biosciences </td><td> 352.340 (40 micromolar) 352 350 (70μm) </td></tr><tr><td> Busulfano </td><td> Sigma </td><td> B2635 </td></tr><tr><td> De pared delgada capilares de vidrio </td><td> Mundial de instrumentos de precisión </td><td> TW 100-3 </td></tr><tr><td> BD intramedic tubo plyethylene (PE100) </td><td> BD </td><td> CA-63018-725 </td></tr><tr><td> Ethicon 6-0 sutura de seda </td><td> Ethicon </td><td> 706G </td></tr><tr><td> Clips de la herida </td><td> BD </td><td> 427631 </td></tr><tr><td> Sigmacote </td><td> Sigma </td><td> SL2 </td></tr><tr><td> X-gal </td><td> Sigma </td><td> B4252 </td></tr><tr><td> Ferrocianuro de potasio </td><td> Sigma </td><td> P9387 </td></tr><tr><td> Ferricianuro de potasio </td><td> Sigma </td><td> P3667 </td></tr><tr><td> cloruro de magnesio </td><td> Sigma </td><td> 208337 </td></tr><tr><td> sodio desoxicolato </td><td> Sigma </td><td> D6750 </td></tr><tr><td> N, N-Dimetilformamida </td><td> Sigma </td><td> D4551 </td></tr><tr><td> Igepal CA-630 </td><td> Sigma </td><td> 18896 </td></tr></tbody></table>

germ cell transplantation and testis tissue xenografting in mice

El transplante de células germinales fue desarrollado por el Dr. Ralph Brinster y sus colegas de la Universidad de Pennsylvania en 1994 1,2. Estos estudios innovadores mostraron que la microinyección de células germinales de los ratones donantes fértiles en los túbulos seminíferos de ratones infértiles receptores de donantes en los resultados derivados de la espermatogénesis y la producción de esperma por parte del animal receptor 2. El uso de machos donantes que transporten la bacteriana β-galactosidasa la identificación de genes permitido de donante-deriva la espermatogénesis y la transmisión de la haplotipo donante a la descendencia por 1 los animales receptores. Sorprendentemente, después de un trasplante en el lumen de los túbulos seminíferos, las células germinales trasplantados fueron capaces de pasar del compartimento luminal de la membrana basal, donde se encuentran las espermatogonias 3. En general se acepta que las ESC sólo son capaces de colonizar el nicho y la espermatogénesis volver a establecerse en los testículos receptores. Por lo tanto, de células germinalesel trasplante ofrece un enfoque funcional para estudiar el nicho de células madre en los testículos y caracterizar supuestas células madre de espermatogonias. Hasta la fecha, el trasplante de células germinales se utiliza para dilucidar la biología básica de células madre, para producir animales transgénicos a través de la manipulación genética de células germinales antes del trasplante 4,5, para estudiar células de Sertoli-germen de interacción celular 6,7, SSC homing y la colonización 3, 8, así como la CSE auto-renovación y diferenciación 9,10. El trasplante de células germinales es también factible en especies de gran tamaño 11. En éstas, las principales aplicaciones son la preservación de la fertilidad, la difusión de la genética de élite en las poblaciones animales, y la generación de animales transgénicos como el estudio de la espermatogénesis y la biología de piel a piel con esta técnica es logísticamente más difícil y caro que en los roedores. El trasplante de células germinales de especies de gran tamaño en los túbulos seminíferos de ratones resulta en colonizatde iones de las células del donante y la expansión espermatogonias, pero no en su diferenciación completa presumiblemente debido a la incompatibilidad del compartimiento célula receptora somática con las células germinales de especies filogenéticamente distantes 12. Un enfoque alternativo es el trasplante de las células germinales de especies de gran tamaño, junto con su entorno compartimento somática. Estamos por primera vez en 2002, que pequeños fragmentos de tejido testicular de los machos inmaduros trasplantadas bajo la piel dorsal de ratones inmunodeficientes son capaces de sobrevivir y tener un desarrollo completo con la producción de esperma fertilización competente 13. Desde entonces, tejido testicular xenoinjerto se ha demostrado para tener éxito en muchas especies y ha surgido como una alternativa valiosa para estudiar el desarrollo testicular y la espermatogénesis de los animales grandes en ratones 14.

Watch this Scientific Journal Video about Germ Cell Transplantation and Testis Tissue Xenografting in Mice at JoVE.com

Germ Cell Transplantation and Testis Tissue Xenografting in Mice

Los protocolos para el trasplante de células germinales de testículo y el xenotrasplante de tejido se describen. Germen de los resultados de trasplante de células en donantes derivado de la espermatogénesis en los testículos receptores y representa un ensayo de reconstitución funcional para la identificación de células madre de espermatogonias (SSC). Tejido testicular xenoinjerto reproduce desarrollo de los testículos y la espermatogénesis de las especies de los distintos donantes en los ratones receptores.

Germen de Trasplante de Células y Tejidos Testículos xenotrasplante en ratones

Germ cell transplantation was developed by Dr. Ralph Brinster and colleagues at the University of Pennsylvania in 19941,2. These ground-breaking studies ...

germ-cell-transplantation-and-testis-tissue-xenografting-in-mice

Research

JoVE Journal

Biology

29.6K vistas.  University of Calgary. Los protocolos para el trasplante de células germinales de testículo y el xenotrasplante de tejido se describen. Germen de los resultados de trasplante de células en donantes derivado de la espermatogénesis en los testículos receptores y representa un ensayo de reconstitución funcional para la identificación de células madre de espermatogonias (SSC). Tejido testicular xenoinjerto reproduce desarrollo de los testículos y la espermatogénesis de las especies de los...

Video: Germ Cell Transplantation and Testis Tissue Xenografting in Mice

Transplantation of Whole Kidney Marrow in Adult Zebrafish

Hematopoietic stem cells (HSC) are a rare population of pluripotent cells that maintain all the differentiated blood lineages throughout the life of an organism. The functional definition of a HSC is a transplanted cell that has the ability to reconstitute all the blood lineages of an irradiated recipient long term. This designation was established by decades of seminal work in mammalian systems. Using hematopoietic cell transplantation (HCT) and reverse genetic manipulations in the mouse, the underlying regulatory factors of HSC biology are beginning to be unveiled, but are still largely under-explored. Recently, the zebrafish has emerged as a powerful genetic model to study vertebrate hematopoiesis. Establishing HCT in zebrafish will allow scientists to utilize the large-scale genetic and chemical screening methodologies available in zebrafish to reveal novel mechanisms underlying HSC regulation. In this article, we demonstrate a method to perform HCT in adult zebrafish. We show the dissection and preparation of zebrafish whole kidney marrow, the site of adult hematopoiesis in the zebrafish, and the introduction of these donor cells into the circulation of irradiated recipient fish via intracardiac injection. Additionally, we describe the post-transplant care of fish in an "ICU" to increase their long-term health. In general, gentle care of the fish before, during, and after the transplant is critical to increase the number of fish that will survive more than one month following the procedure, which is essential for assessment of long term (&lt;3 month) engraftment. The experimental data used to establish this protocol will be published elsewhere. The establishment of this protocol will allow for the merger of large-scale zebrafish genetics and transplant biology.

In this article, we demonstrate a method to perform HCT in adult zebrafish.

El trasplante de médula del riñón entero en adultos de pez cebra

Hematopoietic stem cells (HSC) are a rare population of pluripotent cells that maintain all the differentiated blood lineages throughout the life of an ...

Investigación

Biología

Heterotopic Heart Transplantation in Mice

The mouse heterotopic heart transplantation has been used widely since it was introduced by Drs. Corry and Russell in 1973. It is particularly valuable for studying rejection and immune response now that newer transgenic and gene knockout mice are available, and a large number of immunologic reagents have been developed. The heart transplant model is less stringent than the skin transplant models, although technically more challenging. We have developed a modified technique and have completed over 1000 successful cases of heterotopic heart transplantation in mice. When making anastomosis of the ascending aorta and abdominal aorta, two stay sutures are placed at the proximal and distal apexes of recipient abdominal aorta with the donor s ascending aorta, then using 11-0 suture for anastomosis on both side of aorta with continuing sutures. The stay sutures make the anastomosis easier and 11-0 is an ideal suture size to avoid bleeding and thrombosis.
When making anastomosis of pulmonary artery and inferior vena cava, two stay sutures are made at the proximal apex and distal apex of the recipient s inferior vena cava with the donor s pulmonary artery. The left wall of the inferior vena cava and donor s pulmonary artery is closed with continuing sutures in the inside of the inferior vena cava after, one knot with the proximal apex stay suture the right wall of the inferior vena cava and the donor s pulmonary artery are closed with continuing sutures outside the inferior vena cave with 10-0 sutures. This method is easier to perform because anastomosis is made just on the one side of the inferior vena cava and 10-0 sutures is the right size to avoid bleeding and thrombosis. In this article, we provide details of the technique to supplement the video.

The mouse heterotopic heart transplantation model has been proven by many investigators to be an important method for studying mechanisms of rejection and immune response. However, the techniques involved are still challenging. By modifying standard techniques we have had success with more than 1000 transplants.

El trasplante cardíaco heterotópico en ratones

The mouse heterotopic heart transplantation has been used widely since it was introduced by Drs. Corry and Russell in 1973. It is particularly valuable ...

Small Bowel Transplantation In Mice

Since 1990, the development of tacrolimus-based immunosuppression and improved surgical techniques, the increased array of potent immunosuppressive medications, infection prophylaxis, and suitable patient selection helped improve actuarial graft and patient survival rates for all types of intestine transplantation. Patients with irreversible intestinal failure and complications of parenteral nutrition should now be routinely considered for small intestine transplantation. However, Survival rates for small intestinal transplantation have been slow to improve compares increasingly favorably with renal, liver, heart and lung. The small bowel transplantation is still unsatisfactory compared with other organs. Further progress may depend on better understanding of immunology and physiology of the graft and can be greatly facilitated by animal models. A wider use of mouse small bowel transplantation model is needed in the study of immunology and physiology of the transplantation gut as well as efficient methods in diagnosing early rejection. However, this model is limited to use because the techniques involved is an extremely technically challenging. We have developed a modified technique. When making anastomosis of portal vein and inferior vena cava, two stay sutures are made at the proximal apex and distal apex of the recipient s inferior vena cava with the donor s portal vein. The left wall of the inferior vena cava and donor s portal vein is closed with continuing sutures in the inside of the inferior vena cava after, after one knot with the proximal apex stay suture the right wall of the inferior vena cava and the donor s portal vein are closed with continuing sutures outside the inferior vena cave with 10-0 sutures. This method is easier to perform because anastomosis is made just on the one side of the inferior vena cava and 10-0 sutures is the right size to avoid bleeding and thrombosis.  In this article, we provide details of the technique to supplement the video.

The mouse small bowel transplantation model has been recognized as an important tool to study mechanismes of immune rejection and screen new immunosuppressive drugs. However, this model is limited to use because the techniques involved is an extremely technically challenge. Now we introduce the modified technique.

Trasplante de intestino delgado en ratones

Since 1990, the development of tacrolimus-based immunosuppression and improved surgical techniques, the increased array of potent immunosuppressive ...

Actin Co-Sedimentation Assay; for the Analysis of Protein Binding to F-Actin

The actin cytoskeleton within the cell is a network of actin filaments that allows the movement of cells and cellular processes, and that generates tension and helps maintains cellular shape.  Although the actin cytoskeleton is a rigid structure, it is a dynamic structure that is constantly remodeling.  A number of proteins can bind to the actin cytoskeleton.  The binding of a particular protein to F-actin is often desired to support cell biological observations or to further understand dynamic processes due to remodeling of the actin cytoskeleton. The actin co-sedimentation assay is an in vitro assay routinely used to analyze the binding of specific proteins or protein domains with F-actin.  The basic principles of the assay involve an incubation of the protein of interest (full length or domain of) with F-actin, ultracentrifugation step to pellet F-actin and analysis of the protein co-sedimenting with F-actin.  Actin co-sedimentation assays can be designed accordingly to measure actin binding affinities and in competition assays.

Proteins bind to filamentous actin (F-actin) through distinct actin binding modules. In this video we demonstrate the procedure of actin co-sedimentation, which is an in vitro assay routinely used to analyze proteins or specific domains that bind F-actin.

Actina Co-sedimentación de ensayo, pues el análisis de proteína de unión a la F-actina

The actin cytoskeleton within the cell is a network of actin filaments that allows the movement of cells and cellular processes, and that generates ...

Generation of Human CD40-activated B cells

CD40-activated B cells (CD40-B cells) have been identified as an alternative source of immuno-stimulatory antigen-presenting cells (APC) for cancer immunotherapy 1-3. Compared to Dendritic cells (DCs), the best characterized APC, CD40-B cells have several distinct biological and technical properties. Similar to DCs, B cells show an increased expression of MHC and co-stimulatory molecules (Fig.1b), exhibit a strong migratory capacity and present antigen presentation efficiently to T cells, after stimulation with interleukin-4 and CD40 ligand (CD40L). However, in contrast to immature or mature DCs, CD40-B cells express the full lymph node homing triad consisting of CD62L, CCR7/CXCR4, and leukocyte function antigen-1 (LFA1, CD11a/CD18), necessary for homing to secondary lymphoid organs (Fig.1a) 3. CD40-B cells can be generated without difficulties from very small amounts of peripheral blood which can be further expanded in vitro to very large amounts of highly-pure CD40-B cells (&gt;109 cells per patient) from healthy donors as well as cancer patients (Fig.1c,d) 1,4. 
In this protocol we demonstrate how to obtain fully activated CD40-B cells from human PBMC. Key molecules for the cell culture are CD40 ligand, interleukin-4 (IL-4) and cyclosporin A (CsA), which are replenished in a 3-4 day culture cycle. For laboratory purposes CD40-stimulation is provided by NIH/3T3 cells expressing recombinant human CD40 ligand (tCD40L NIH/3T3) 5. To avoid contamination with non-transfected cells, expression of the human CD40 ligand on the transfectants has to be checked regularly (Fig.2). 
After 14 days CD40-B cell cultures consist of more than 95% pure B cells and an expansion of CD40-B cells over 65 days is frequently possible without any loss of function 1, 4. CD40-B cells efficiently take up, process and present antigens to T cells 6. They do not only prime na&#970;ve, but also expand memory T cells 7,8. CD40-activated B cells can be used to study B-cell activation, differentiation and function. Moreover, they represent a promising tool for therapeutic or preventive vaccination against tumors 9.

In this video we present the ex vivo generation and expansion of human CD40-activated B cells (CD40-B) from peripheral blood mononuclear cells (PBMC) by stimulation with CD40 ligand and interleukin-4.

Generación de humanos CD40 activado por células B

CD40-activated B cells (CD40-B cells) have been identified as an alternative source of immuno-stimulatory antigen-presenting cells (APC) for cancer ...

Tissue Targeted Embryonic Chimeras: Zebrafish Gastrula Cell Transplantation

Certain fundamental questions in the field of developmental biology can only be answered when cells are placed in novel environments or when small groups of cells in a larger context are altered.  Watching how one cell interacts with and behaves in a unique environment is essential to characterizing cell functions.  Determining how the localized misexpression of a specific protein influences surrounding cells provides insightful information on the roles that protein plays in a variety of developmental processes.  Our lab uses the zebrafish model system to uniquely combine genetic approaches with classical transplantation techniques to generate genotypic or phenotypic chimeras.  We study neuron-glial cell interactions during the formation of forebrain commissures in zebrafish. This video describes a method that allows our lab to investigate the role of astroglial populations in the diencephalon and the roles of specific guidance cues that influence projecting axons as they cross the midline. Due to their transparency zebrafish embryos are ideal models for this type of ectopic cell placement or localized gene misexpression. Tracking transplanted cells can be accomplished using a vital dye or a transgenic fish line expressing a fluorescent protein. We demonstrate here how to prepare donor embryos with a vital dye tracer for transplantation, as well as how to extract and transplant cells from one gastrula staged embryo to another.  We present data showing ectopic GFP+ transgenic cells within the forebrain of zebrafish embryos and characterize the location of these cells with respect to forebrain commissures.  In addition, we show laser scanning confocal timelapse microscopy of Alexa 594 labeled cells transplanted into a GFP+ transgenic host embryo.  These data provide evidence that gastrula staged transplantation enables the targeted positioning of ectopic cells to address a variety of questions in Developmental Biology.

Zebrafish cell transplantation enables the combination of genetics and embryology to generate tissue specific chimeras. This video demonstrates gastrula staged cell transplantations that have allowed our lab to investigate the roles of astroglial populations and specific guidance cues during commissure formation in the forebrain.

Tejido diana quimeras embrionarias: pez cebra Gástrula Trasplante de células

Certain fundamental questions in the field of developmental biology can only be answered when cells are placed in novel environments or when small groups ...

Heterotopic and Orthotopic Tracheal Transplantation in Mice used as Models to Study the Development of  Obliterative Airway Disease

Obliterative airway disease (OAD) is the major complication after lung transplantations that limits long term survival (1-7).
To study the pathophysiology, treatment and prevention of OAD, different animal models of tracheal transplantation in rodents have been developed (1-7). Here, we use two established models of trachea transplantation, the heterotopic and orthotopic model and demonstrate their advantages and limitations.
For the heterotopic model, the donor trachea is wrapped into the greater omentum of the recipient, whereas the donor trachea is anastomosed by end-to-end anastomosis in the orthotopic model.
In both models, the development of obliterative lesions histological similar to clinical OAD has been demonstrated (1-7).
This video shows how to perform both, the heterotopic as well as the orthotopic tracheal transplantation technique in mice, and compares the time course of OAD development in both models using histology.

This video shows and compares two experimental models to study the development of obliterative airway disease (OAD) in mice, the heterotopic and orthotopic tracheal transplantation model.

El trasplante heterotópico traqueal y ortotópico en ratones utilizados como modelos para estudiar el desarrollo de la enfermedad de las vías respiratorias obliterante

Obliterative airway disease (OAD) is the major complication after lung transplantations that limits long term survival (1-7).
To study the ...

Planarian Immobilization, Partial Irradiation, and Tissue Transplantation

The planarian, a freshwater flatworm, has proven to be a powerful system for dissecting metazoan regeneration and stem cell biology1,2. Planarian regeneration of any missing or damaged tissues is made possible by adult stem cells termed neoblasts3. Although these stem cells have been definitively shown to be pluripotent and singularly capable of reconstituting an entire animal4, the heterogeneity within the stem cell population and the dynamics of their cellular behaviors remain largely unresolved. Due to the large number and wide distribution of stem cells throughout the planarian body plan, advanced methods for manipulating subpopulations of stem cells for molecular and functional study in vivo are needed.
Tissue transplantation and partial irradiation are two methods by which a subpopulation of planarian stem cells can be isolated for further study. Each technique has distinct advantages. Tissue transplantation allows for the introduction of stem cells, into a na&iuml;ve host, that are either inherently genetically distinct or have been previously treated pharmacologically. Alternatively, partial irradiation allows for the isolation of stem cells within a host, juxtaposed to tissue devoid of stem cells, without the introduction of a wound or any breech in tissue integrity. Using these two methods, one can investigate the cell autonomous and non-autonomous factors that control stem cell functions, such as proliferation, differentiation, and migration.
Both tissue transplantation5,6 and partial irradiation7 have been used historically in defining many of the questions about planarian regeneration that remain under study today. However, these techniques have remained underused due to the laborious and inconsistent nature of previous methods. The protocols presented here represent a large step forward in decreasing the time and effort necessary to reproducibly generate large numbers of grafted or partially irradiated animals with efficacies approaching 100 percent. We cover the culture of large animals, immobilization, preparation for partial irradiation, tissue transplantation, and the optimization of animal recovery. Furthermore, the work described here demonstrates the first application of the partial irradiation method for use with the most widely studied planarian, Schmidtea mediterranea. Additionally, efficient tissue grafting in planaria opens the door for the functional testing of subpopulations of na&iuml;ve or treated stem cells in repopulation assays, which has long been the gold-standard method of assaying adult stem cell potential in mammals8. Broad adoption of these techniques will no doubt lead to a better understanding of the cellular behaviors of adult stem cells during tissue homeostasis and regeneration.

An effective method for grafting tissue of defined and consistent size between planaria is described. Also included is a description of how the immobilization technique used for transplantation can be adapted, in conjunction with lead shields, for the partial irradiation of live animals.

La inmovilización de las planarias, irradiación parcial, y el trasplante de tejido

The planarian, a freshwater flatworm, has proven to be a powerful system for dissecting metazoan regeneration and stem cell biology1,2. Planarian ...

Studying Proteolysis of Cyclin B at the Single Cell Level in Whole Cell Populations

Equal distribution of chromosomes between the two daughter cells during cell division is a prerequisite for guaranteeing genetic stability 1. Inaccuracies during chromosome separation are a hallmark of malignancy and associated with progressive disease 2-4. The spindle assembly checkpoint (SAC) is a mitotic surveillance mechanism that holds back cells at metaphase until every single chromosome has established a stable bipolar attachment to the mitotic spindle1. The SAC exerts its function by interference with the activating APC/C subunit Cdc20 to block proteolysis of securin and cyclin B and thus chromosome separation and mitotic exit. Improper attachment of chromosomes prevents silencing of SAC signaling and causes continued inhibition of APC/CCdc20 until the problem is solved to avoid chromosome missegregation, aneuploidy and malignant growths1.
Most studies that addressed the influence of improper chromosomal attachment on APC/C-dependent proteolysis took advantage of spindle disruption using depolymerizing or microtubule-stabilizing drugs to interfere with chromosomal attachment to microtubules. Since interference with microtubule kinetics can affect the transport and localization of critical regulators, these procedures bear a risk of inducing artificial effects 5.
To study how the SAC interferes with APC/C-dependent proteolysis of cyclin B during mitosis in unperturbed cell populations, we established a histone H2-GFP-based system which allowed the simultaneous monitoring of metaphase alignment of mitotic chromosomes and proteolysis of cyclin B 6.
To depict proteolytic profiles, we generated a chimeric cyclin B reporter molecule with a C-terminal SNAP moiety 6 (Figure 1). In a self-labeling reaction, the SNAP-moiety is able to form covalent bonds with alkylguanine-carriers (SNAP substrate) 7,8 (Figure 1). SNAP substrate molecules are readily available and carry a broad spectrum of different fluorochromes. Chimeric cyclin B-SNAP molecules become labeled upon addition of the membrane-permeable SNAP substrate to the growth medium 7 (Figure 1). Following the labeling reaction, the cyclin B-SNAP fluorescence intensity drops in a pulse-chase reaction-like manner and fluorescence intensities reflect levels of cyclin B degradation 6 (Figure 1). Our system facilitates the monitoring of mitotic APC/C-dependent proteolysis in large numbers of cells (or several cell populations) in parallel. Thereby, the system may be a valuable tool to identify agents/small molecules that are able to interfere with proteolytic activity at the metaphase to anaphase transition. Moreover, as synthesis of cyclin B during mitosis has recently been suggested as an important mechanism in fostering a mitotic block in mice and humans by keeping cyclin B expression levels stable 9,10, this system enabled us to analyze cyclin B proteolysis as one element of a balanced equilibrium 6.

Metaphase to anaphase transition is triggered through anaphase-promoting complex (APC/C)-dependent ubiquitination and subsequent destruction of cyclin B. Here, we established a system which, following pulse-chase labeling, allows monitoring cyclin B proteolysis in entire cell populations and facilitates the detection of interference by the mitotic checkpoint.

El estudio de la proteólisis de ciclina B en el nivel de células individuales en poblaciones de células enteras

Equal distribution of chromosomes between the two daughter cells during cell division is a prerequisite for guaranteeing genetic stability 1. Inaccuracies ...

Separation of Spermatogenic Cell Types Using STA-PUT Velocity Sedimentation

Mammalian spermatogenesis is a complex differentiation process that occurs in several stages in the seminiferous tubules of the testes. Currently, there is no reliable cell culture system allowing for spermatogenic differentiation in vitro, and most biological studies of spermatogenic cells require tissue harvest from animal models like the mouse and rat. Because the testis contains numerous cell types - both non-spermatogenic (Leydig, Sertoli, myeloid, and epithelial cells) and spermatogenic (spermatogonia, spermatocytes, round spermatids, condensing spermatids and spermatozoa) - studies of the biological mechanisms involved in spermatogenesis require the isolation and enrichment of these different cell types. The STA-PUT method allows for the separation of a heterogeneous population of cells - in this case, from the testes - through a linear BSA gradient. Individual cell types sediment with different sedimentation velocity according to cell size, and fractions enriched for different cell types can be collected and utilized in further analyses. While the STA-PUT method does not result in highly pure fractions of cell types, e.g. as can be obtained with certain cell sorting methods, it does provide a much higher yield of total cells in each fraction (~1 x 108 cells/spermatogenic cell type from a starting population of 7-8 x 108 cells). This high yield method requires only specialized glassware and can be performed in any cold room or large refrigerator, making it an ideal method for labs that have limited access to specialized equipment like a fluorescence activated cell sorter (FACS) or elutriator.

The STA-PUT method allows for the separation of different populations of spermatogenic cells based on size and density.

La separación de los tipos de células de espermatogénesis Usando STA-PUT Sedimentación Velocity

Mammalian spermatogenesis is a complex  differentiation process that occurs in several stages in the seminiferous  tubules of the testes. Currently, there ...