Los autores agradecen a Christiane Pahrmann (Lab Manager).

<ol><li> Mujer ratones Balb / C (8-12 semanas) son adquiridos de Charles River Laboratories (Sulzfeld, Alemania). Los ratones se encuentran en condiciones convencionales, alimentados con comida estándar de ratones y agua ad libitum. </li><li> 2% Isofluran se utiliza para la anestesia. </li></ol> PREPARACIÓN DE LOS DONANTES <ol><li> Afeitar el vello abdominal y desinfectar el área con betaisodona. </li><li> Bajo visión microscópica, realizar una incisión cervical desde el nivel de la laringe con el esternón. </li><li> Quitar la grasa subcutánea y los músculos de la correa para obtener una visión clara de la tráquea. </li><li> Disecar la tráquea de los tejidos circundantes, como el esófago, los nervios, arterias y tejido conectivo. </li><li> Retire toda la tráquea (desde la laringe hasta la bifurcación). </li><li> Enjuague bien el trasplante con una solución salina fría y guardar el injerto a 4 ° C. </li><li> El donante es la eutanasia por dislocación cervical después de la cosecha de la tráquea. </li></ol> BENEFICIARIO: trasplante heterotópico <ol><li> Afeitar el vello abdominal en un amplio margen alrededor de la incisión y desinfectar la zona tres veces con betaisodona (betadine), seguido por el alcohol. Los ojos deben ser lubricados con un producto de ungüento oftálmico para evitar que las córneas que se sequen. </li><li> Realizar una laparotomía media y el lugar en el intestino en un guante estéril y humectada. </li><li> Difundir el epiplón mayor cuidado. Coloque el injerto en el centro y fijar con una sola sutura con 8-0 (Prolene, Ethicon, Alemania). </li><li> Cubren totalmente el trasplante con el epiplón mayor y fijar el injerto con una sola sutura 8-0 (Prolene, Ethicon, Alemania). </li><li> Reubicar a los intestinos en el abdomen y lave con solución salina tibia y estéril antes del cierre. </li><li> Cerca de dos capas - pared abdominal y la capa de la piel con un patrón continuo con prolene 7-0 para el músculo y Vicryl 7-0 para la piel. </li></ol> BENEFICIARIO: trasplante ortotópico <ol><li> Afeitar el vello abdominal en un amplio margen alrededor de la incisión y desinfectar la zona tres veces con betaisodona (betadine), seguido por el alcohol. Los ojos deben ser lubricados con un producto de ungüento oftálmico para evitar que las córneas que se sequen. </li><li> Divide los músculos correa para visualizar el complejo laringotraqueal completa. </li><li> Diseccionar cuidadosamente la tráquea de los tejidos circundantes, tenga cuidado para preservar los nervios laríngeos recurrentes. </li><li> Divide la tráquea tres anillos de caudal desde el cricoides. El animal mantiene la respiración fisiológica a través de la traqueotomía. </li><li> Asegúrese de limpiar los bordes de la tráquea en el receptor, así como en el injerto. </li><li> El injerto se interpone entre el receptor defectos traqueal y orientado a mantener la polaridad anatómica. </li><li> Utilizando 8-0 (Prolene, Ethicon, Alemania) anastomosis del injerto donante con la parte distal (mediastino) tráquea. La cara posterior de la anastomosis se realiza en forma continua en funcionamiento. La cara anterior se completa con puntos de sutura interrumpida. </li><li> Eliminar las secreciones de las vías respiratorias. </li><li> La anastomosis proximal se completa en la misma forma que la distal. </li><li> Asegurar la integridad de las vías respiratorias y la respiración adecuada y espontánea. </li><li> La ubicación de la correa de los músculos y cerrar el tejido subcutáneo y la capa de la piel con suturas 6-0 (Vicryl, Ethicon, Alemania) con un patrón continuo. </li><li> El uso de anestesia por inyección para el destinatario, por lo tanto, el animal mantendrá la respiración fisiológica a través de la tráquea. Una combinación de 75/1/0.2 mg / kg de propofol, medetomidina y fentanilo, respectivamente, se utiliza para la anestesia ip en ratones. </li></ol><img src="/files/ftp_upload/1437/1437Fig1.jpg" alt="Figura 1" /> Figura 1: donantes tráquea. 1A: Donantes tráquea in situ después de la preparación. 1B: tráquea extirpada de los donantes. 1C: Donantes tráquea después de la explantación. <img src="/files/ftp_upload/1437/1437Fig2.jpg" alt="Figura 2" /> Figura 2: Modelo heterotópico. 2A: El injerto se coloca en el centro del epiplón mayor. 2B: El injerto se fija en ambos extremos con una sutura simple. 2C: El injerto se envuelve en el epiplón mayor y se fija con una sutura simple. <img src="/files/ftp_upload/1437/1437Fig3.jpg" alt="Figura 3" /> Figura 3: Modelo ortotópico. 3A: El injerto se interpone entre el receptor defectos traqueal y la pared posterior se anastomosa en forma continua en funcionamiento. 3B: La pared anterior se completa con una sola sutura. <img src="/files/ftp_upload/1437/1437Fig4.jpg" alt="Figura 4" /> Figura 4: Histología. 4A: Reformado tráquea trasplantada heterotópico después de 28 días en la estación H + Enando (15x). Tenga en cuenta la destrucción del 100% luminal. 4B: Reformado tráquea trasplantada ortotópico después de 60 días en la tinción H + E (15x). Obliteración luminal máximo alcanzado es de aproximadamente 45%. <table border="1"><tbody><tr><td></td><td> Modelo heterotópico trasplante traqueal </td><td> Modelo ortotópico de trasplante traqueal </td></tr><tr><td> Ventajas </td><td> + Fácil para llevar a cabo + Obliteración luminal con oclusión completa las vías respiratorias después de 28 días + No hay afecto físico de los animales por el OAD </td><td> + Física ventilación del injerto + Administración de fármacos inhalados posible + Fuertes reacciones inmunológicas como la producción de anticuerpos IgM aloreactivas + Torácica medio fisiológico + Traqueal-traqueal anastomosis imita a la práctica clínica </td></tr><tr><td> Desventajas </td><td> - No había ventilación de la tráquea trasplantada - No hay evaluación de patógenos inhalados posible - La inhibición de la depuración mucociliar y retención de secreciones - Microambiente peritoneal en lugar de medio torácica </td><td> - Formación necesaria quirúrgica - Obliteración luminal con aplicación de la oclusión luminal. 45% después de 60 días - Los animales pueden desarrollar síntomas de la OAD </td></tr></tbody></table> Tabla 1: Ventajas y desventajas del trasplante heterotópico traqueal y ortotópico.

<ol>
	<li>Adams, B., Berry, G., Huang, X., Shorthouse, R., Brazelton, T., Morris, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunosuppressive+therapies+for+the+prevention+and+treatment+of+obliterative+airway+disease+in+heterotopic+rat+trachea+allografts.">Immunosuppressive therapies for the prevention and treatment of obliterative airway disease in heterotopic rat trachea allografts.</a> Transplantation. 69, 2260-2266 .</li><li>Adams, B., Brazelton, T., Berry, G., Morris, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+respiratory+epithelium+in+a+rat+model+of+obliterative+airway+disease.">The role of respiratory epithelium in a rat model of obliterative airway disease.</a> Transplantation. 69, 661-665 .</li><li>Deuse, T., Schrepfer, S., Reichenspurner, H., Hoyt, G., Fischbein, M., Robbins, R., Pelletier, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Techniques+for+experimental+heterotopic+and+orthotopic+tracheal+transplantations+&amp;#x2013;+when+to+use+which+model;+Transplant+Immunology+17.">Techniques for experimental heterotopic and orthotopic tracheal transplantations &amp;#x2013; when to use which model; Transplant Immunology 17.</a> , 255-261 (2007).</li><li>Hele, D., Yacoub, M., Belvisi, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+heterotopic+tracheal+allograft+as+an+animal+model+of+obliterative+bronchiolitis.">The heterotopic tracheal allograft as an animal model of obliterative bronchiolitis.</a> Respiratory Research. 2, 169-183 .</li><li>Hertz, M., Jessurun, J., King, M., Savic, S., Murray, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reproduction+of+the+obliterative+bronchiolitis+lesion+after+heterotopic+transplantation+of+mouse+airways.">Reproduction of the obliterative bronchiolitis lesion after heterotopic transplantation of mouse airways.</a> American Journal of Pathology. 142, (1993).</li><li>McDyer, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+and+murine+obliterative+bronchiolitis+in+transplant.">Human and murine obliterative bronchiolitis in transplant.</a> Proceedings of the American Thoracic Society. 4, 37-43 .</li><li>Schrepfer, S., Deuse, T., Hoyt, G., Sheikh, A., Hoffmann, J., Reichenspurner, H., Robbins, R., Pelletier, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Experimental+orthotopic+tracheal+transplantation:+the+stanford+technique.">Experimental orthotopic tracheal transplantation: the stanford technique.</a> Microsurgery. 27, 187-189 (2007).</li></ol>

Sonja Schrepfer ha recibido una beca de investigación de la Deutsche Forschungsgemeinschaft (DFG) (SCHR992/3-1).] Todos los animales recibieron cuidados humanos de conformidad con los Principios de Cuidado de Animales de laboratorio, los experimentos con animales se realizaron de acuerdo con las directrices y normas establecidas por la Sociedad Nacional de Investigaciones Médicas y la Guía para el Cuidado y Uso de Animales de Laboratorio publicado por la Institutos Nacionales de Salud (National Institutes of Health publicación 85-23, revisado 1985). Todos los animales se obtuvieron de Charles River Laboratories (Sulzfeld, Alemania) y se mantuvieron en las instalaciones de cuidado de los animales del Hospital Universitario de Eppendorf en Hamburgo. Los animales recibieron comida estándar y agua ad libitum.

Los ratones están disponibles en diferentes transgeneic y el modelo de octavos de final, y por lo tanto, adecuado para estudiar las cuestiones relacionadas con la mecánica OAD (4). Ambos modelos de trasplante de tráquea se muestra en este video se puede utilizar como modelos confiables para estudiar el desarrollo OAD. Sin embargo, cada modelo muestra las ventajas y limitaciones. El trasplante traqueal heterotópico es fácil de realizar y no requiere de entr...

heterotopic and orthotopic tracheal transplantation in mice used as models to study the development of  obliterative airway disease

Obliterante las vías respiratorias enfermedad (OAD) es la complicación más importante después de un trasplante de pulmón que los límites de la supervivencia a largo plazo (1-7). Para el estudio de la fisiopatología, tratamiento y prevención de los modelos de OAD, animales diferentes de trasplante de tráquea en los roedores se han desarrollado (1-7). Aquí, hemos utilizado dos modelos establecidos de transplante de tráquea, el modelo ortotópico y heterotópico y demostrar sus ventajas y limitaciones. Para el modelo heterotópico, la tráquea del donante se envuelve en el epiplón mayor del receptor, mientras que el donante la tráquea se anastomosa por anastomosis término-a-extremo en el modelo ortotópico. En ambos modelos, el desarrollo de lesiones histológicas similares a obliterante OAD clínicos se ha demostrado (1-7). Este vídeo muestra cómo llevar a cabo tanto, el heterotópico, así como la técnica de trasplante ortotópico traqueal en ratones, y se compara la evolución temporal del desarrollo de AOD en ambos modelos con la histología.

Watch this Scientific Journal Video about Heterotopic and Orthotopic Tracheal Transplantation in Mice used as Models to Study the Development of  Obliterative Airway Disease at JoVE.com

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

Este video muestra y se comparan dos modelos experimentales para estudiar el desarrollo de la enfermedad de las vías respiratorias obliterante (OAD) en ratones, el modelo de trasplante heterotópico traqueal y ortotópico.

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 ...

heterotopic-orthotopic-tracheal-transplantation-mice-used-as-models

Research

JoVE Journal

Biology

13.8K vistas.  University Heart Center Hamburg. Este video muestra y se comparan dos modelos experimentales para estudiar el desarrollo de la enfermedad de las vías respiratorias obliterante (OAD) en ratones, el modelo de trasplante heterotópico traqueal y ortotópico.

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

Testing Visual Sensitivity to the Speed and Direction of Motion in Lizards

Testing visual sensitivity in any species provides basic information regarding behaviour, evolution, and ecology. However, testing specific features of the visual system provide more empirical evidence for functional applications. Investigation into the sensory system provides information about the sensory capacity, learning and memory ability, and establishes known baseline behaviour in which to gauge deviations (Burghardt, 1977). However, unlike mammalian or avian systems, testing for learning and memory in a reptile species is difficult. Furthermore, using an operant paradigm as a psychophysical measure of sensory ability is likewise as difficult. Historically, reptilian species have responded poorly to conditioning trials because of issues related to motivation, physiology, metabolism, and basic biological characteristics. Here, I demonstrate an operant paradigm used a novel model lizard species, the Jacky dragon (Amphibolurus muricatus) and describe how to test peripheral sensitivity to salient speed and motion characteristics. This method uses an innovative approach to assessing learning and sensory capacity in lizards. I employ the use of random-dot kinematograms (RDKs) to measure sensitivity to speed, and manipulate the level of signal strength by changing the proportion of dots moving in a coherent direction. RDKs do not represent a biologically meaningful stimulus, engages the visual system, and is a classic psychophysical tool used to measure sensitivity in humans and other animals. Here, RDKs are displayed to lizards using three video playback systems. Lizards are to select the direction (left or right) in which they perceive dots to be moving. Selection of the appropriate direction is reinforced by biologically important prey stimuli, simulated by computer-animated invertebrates.

Testing visual sensitivity in lizards using an operant conditioning paradigm that employs video playback of random-dot kinematograms and computer-generated invertebrates.

Prueba de sensibilidad visual a la velocidad y dirección del movimiento en lagartos

Testing visual sensitivity in any species provides basic information regarding behaviour, evolution, and ecology. However, testing specific features of ...

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 ...

Quantification of &gamma;H2AX Foci in Response to Ionising Radiation

DNA double-strand breaks (DSBs), which are induced by either endogenous metabolic processes or by exogenous sources, are one of the most critical DNA lesions with respect to survival and preservation of genomic integrity. An early response to the induction of DSBs is phosphorylation of the H2A histone variant, H2AX, at the serine-139 residue, in the highly conserved C-terminal SQEY motif, forming &gamma;H2AX1. Following induction of DSBs, H2AX is rapidly phosphorylated by the phosphatidyl-inosito 3-kinase (PIKK) family of proteins, ataxia telangiectasia mutated (ATM), DNA-protein kinase catalytic subunit and ATM and RAD3-related (ATR)2. Typically, only a few base-pairs (bp) are implicated in a DSB, however, there is significant signal amplification, given the importance of chromatin modifications in DNA damage signalling and repair. Phosphorylation of H2AX mediated predominantly by ATM spreads to adjacent areas of chromatin, affecting approximately 0.03% of total cellular H2AX per DSB2,3. This corresponds to phosphorylation of approximately 2000 H2AX molecules spanning ~2 Mbp regions of chromatin surrounding the site of the DSB and results in the formation of discrete &gamma;H2AX foci which can be easily visualized and quantitated by immunofluorescence microscopy2. The loss of &gamma;H2AX at DSB reflects repair, however, there is some controversy as to what defines complete repair of DSBs; it has been proposed that rejoining of both strands of DNA is adequate however, it has also been suggested that re-instatement of the original chromatin state of compaction is necessary4-8. The disappearence of &gamma;H2AX involves at least in part, dephosphorylation by phosphatases, phosphatase 2A and phosphatase 4C5,6. Further, removal of &gamma;H2AX by redistribution involving histone exchange with H2A.Z has been implicated7,8. Importantly, the quantitative analysis of &gamma;H2AX foci has led to a wide range of applications in medical and nuclear research. Here, we demonstrate the most commonly used immunofluorescence method for evaluation of initial DNA damage by detection and quantitation of &gamma;H2AX foci in &gamma;-irradiated adherent human keratinocytes9.

Quantification of &#947;H2AX Foci in Response to Ionising Radiation

Quantification of DNA double-strand streaks using &gamma;H2AX formation as a molecular marker has become an invaluable tool in radiation biology. Here we demonstrate the use of an immunofluorescence assay for quantification of &gamma;H2AX foci after exposure of cells to radiation.

La cuantificación de γH2AX focos en respuesta a la radiación ionizante

DNA double-strand breaks (DSBs), which are induced by either endogenous metabolic processes or by exogenous sources, are one of the most critical DNA ...

Manual Restraint and Common Compound Administration Routes in Mice and Rats

Being able to safely and effectively restrain mice and rats is an important part of conducting research. Working confidently and humanely with mice and rats requires a basic competency in handling and restraint methods. This article will present the basic principles required to safely handle animals. One-handed, two-handed, and restraint with specially designed restraint objects will be illustrated. Often, another part of the research or testing use of animals is the effective administration of compounds to mice and rats. Although there are a large number of possible administration routes (limited only by the size and organs of the animal), most are not used regularly in research. This video will illustrate several of the more common routes, including intravenous, intramuscular, subcutaneous, and oral gavage. The goal of this article is to expose a viewer unfamiliar with these techniques to basic restraint and substance administration routes. This video does not replace required hands-on training at your facility, but is meant to augment and supplement that training.

Working safely and humanely with research rodents requires a core competency in handling and restraint methods. This article will present the basic principles required to safely handle and effectively administer compounds to mice and rats.

Sujeción manual y de las rutas de la administración del compuesto en ratones y ratas

Being able to safely and effectively restrain mice and rats is an important part of conducting research.  Working confidently and humanely with mice and ...

Evaluation of Mammary Gland Development and Function in Mouse Models

The human mammary gland is composed of 15-20 lobes that secrete milk into a branching duct system opening at the nipple. Those lobes are themselves composed of a number of terminal duct lobular units made of secretory alveoli and converging ducts1. In mice, a similar architecture is observed at pregnancy in which ducts and alveoli are interspersed within the connective tissue stroma. The mouse mammary gland epithelium is a tree like system of ducts composed of two layers of cells, an inner layer of luminal cells surrounded by an outer layer of myoepithelial cells denoted by the confines of a basement membrane2. At birth, only a rudimental ductal tree is present, composed of a primary duct and 15-20 branches. Branch elongation and amplification start at the beginning of puberty, around 4 weeks old, under the influence of hormones3,4,5. At 10 weeks, most of the stroma is invaded by a complex system of ducts that will undergo cycles of branching and regression in each estrous cycle until pregnancy2. At the onset of pregnancy, a second phase of development begins, with the proliferation and differentiation of the epithelium to form grape-shaped milk secretory structures called alveoli6,7. Following parturition and throughout lactation, milk is produced by luminal secretory cells and stored within the lumen of alveoli. Oxytocin release, stimulated by a neural reflex induced by suckling of pups, induces synchronized contractions of the myoepithelial cells around the alveoli and along the ducts, allowing milk to be transported through the ducts to the nipple where it becomes available to the pups 8. Mammary gland development, differentiation and function are tightly orchestrated and require, not only interactions between the stroma and the epithelium, but also between myoepithelial and luminal cells within the epithelium9,10,11. Thereby, mutations in many genes implicated in these interactions may impair either ductal elongation during puberty or alveoli formation during early pregnancy, differentiation during late pregnancy and secretory activation leading to lactation12,13. In this article, we describe how to dissect mouse mammary glands and assess their development using whole mounts. We also demonstrate how to evaluate myoepithelial contractions and milk ejection using an ex-vivo oxytocin-based functional assay. The effect of a gene mutation on mammary gland development and function can thus be determined in situ by performing these two techniques in mutant and wild-type control mice.

This method describes how to dissect and assess mammary gland development and function from mice. Excised mammary glands are assessed for the degree of development using whole mount while milk ejection is evaluated using an oxytocin-based myoepithelial cell contraction assay.

Evaluación de la glándula mamaria de desarrollo y función en modelos de ratón

The human mammary gland is composed of 15-20 lobes that secrete milk into a branching duct system opening at the nipple. Those lobes are themselves ...

Diagnostic Necropsy and Selected Tissue and Sample Collection in Rats and Mice

There are multiple sample types that may be collected from a euthanized animal in order to help diagnose or discover infectious agents in an animal colony. Proper collection of tissues for further histological processing can impact the quality of testing results. This article describes the conduct of a basic gross examination including identification of heart, liver, lungs, kidneys, and spleen, as well as how to collect those organs. Additionally four of the more difficult tissue/sample collection techniques are demonstrated. Lung collection and perfusion can be particularly challenging as the tissue needs to be properly inflated with a fixative in order for inside of the tissue to fix properly and to enable thorough histologic evaluation. This article demonstrates the step by step technique to remove the lung and inflate it with fixative in order to achieve optimal fixation of the tissue within 24 hours. Brain collection can be similarly challenging as the tissue is soft and easily damaged. This article demonstrates the step by step technique to expose and remove the brain from the skull with minimal damage to the tissue. The mesenteric lymph node is a good sample type in which to detect many common infectious agents as enteric viruses persist longer in the lymph node than they are shed in feces. This article demonstrates the step by step procedure for locating and aseptically removing the mesenteric lymph node. Finally, identification of infectious agents of the respiratory tract may be performed by bacterial culture or PCR testing of nasal and/or bronchial fluid aspirates taken at necropsy. This procedure describes obtaining and preparing the respiratory aspirate sample for bacterial culture and PCR testing.

This article describes the procedures for conducting a basic postmortem examination of a mouse or rat, and the collection of basic organs, as well as more challenging sample types from for histological, microbiological, and PCR evaluation.

La necropsia de diagnóstico y selecta colección de tejidos y muestras en ratas y ratones

There are multiple sample types that may be collected from a euthanized animal in order to help diagnose or discover infectious agents in an animal ...

Using SecM Arrest Sequence as a Tool to Isolate Ribosome Bound Polypeptides

Extensive research has provided ample evidences suggesting that protein folding in the cell is a co-translational process1-5. However, the exact pathway that polypeptide chain follows during co-translational folding to achieve its functional form is still an enigma. In order to understand this process and to determine the exact conformation of the co-translational folding intermediates, it is essential to develop techniques that allow the isolation of RNCs carrying nascent chains of predetermined sizes to allow their further structural analysis.
SecM (secretion monitor) is a 170 amino acid E. coli protein that regulates expression of the downstream SecA (secretion driving) ATPase in the secM-secA operon6. Nakatogawa and Ito originally found that a 17 amino acid long sequence (150-FSTPVWISQAQGIRAGP-166) in the C-terminal region of the SecM protein is sufficient and necessary to cause stalling of SecM elongation at Gly165, thereby producing peptidyl-glycyl-tRNA stably bound to the ribosomal P-site7-9. More importantly, it was found that this 17 amino acid long sequence can be fused to the C-terminus of virtually any full-length and/or truncated protein thus allowing the production of RNCs carrying nascent chains of predetermined sizes7. Thus, when fused or inserted into the target protein, SecM stalling sequence produces arrest of the polypeptide chain elongation and generates stable RNCs both in vivo in E. coli cells and in vitro in a cell-free system. Sucrose gradient centrifugation is further utilized to isolate RNCs.
The isolated RNCs can be used to analyze structural and functional features of the co-translational folding intermediates. Recently, this technique has been successfully used to gain insights into the structure of several ribosome bound nascent chains10,11. Here we describe the isolation of bovine Gamma-B Crystallin RNCs fused to SecM and generated in an in vitro translation system.

We describe here a technique that is now routinely used to isolate stably bound ribosome nascent chain complexes (RNCs). This technique takes advantage of the discovery that a 17 amino acid long SecM "arrest sequence" can halt translation elongation in a prokaryotic (E. coli) system, when inserted into (or fused to the C-terminus) of virtually any protein.

Usar secuencias de detención SecM como una herramienta para aislar Ribosome polipéptidos enlazados

Extensive research has provided ample evidences suggesting that protein folding in the cell is a co-translational process1-5. However, the exact pathway ...

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

The folding and assembly of proteins is essential for protein function, the long-term health of the cell, and longevity of the organism. Historically, the function and regulation of protein folding was studied in vitro, in isolated tissue culture cells and in unicellular organisms. Recent studies have uncovered links between protein homeostasis (proteostasis), metabolism, development, aging, and temperature-sensing. These findings have led to the development of new tools for monitoring protein folding in the model metazoan organism Caenorhabditis elegans. In our laboratory, we combine behavioral assays, imaging and biochemical approaches using temperature-sensitive or naturally occurring metastable proteins as sensors of the folding environment to monitor protein misfolding. Behavioral assays that are associated with the misfolding of a specific protein provide a simple and powerful readout for protein folding, allowing for the fast screening of genes and conditions that modulate folding. Likewise, such misfolding can be associated with protein mislocalization in the cell. Monitoring protein localization can, therefore, highlight changes in cellular folding capacity occurring in different tissues, at various stages of development and in the face of changing conditions. Finally, using biochemical tools ex vivo, we can directly monitor protein stability and conformation. Thus, by combining behavioral assays, imaging and biochemical techniques, we are able to monitor protein misfolding at the resolution of the organism, the cell, and the protein, respectively.

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

To study the relationship between protein homeostasis, stress and aging, we monitored changes in protein folding by following protein dysfunction, protein localization in the cell and protein stability at the organismal, cellular and protein levels, using the genetically tractable metazoan Caenorhabditis elegans as a model system.

The folding and assembly of proteins is essential for protein function, the long-term health of the cell, and longevity of the organism. Historically, the ...

Tissue Triage and Freezing for Models of Skeletal Muscle Disease

Skeletal muscle is a unique tissue because of its structure and function, which requires specific protocols for tissue collection to obtain optimal results from functional, cellular, molecular, and pathological evaluations. Due to the subtlety of some pathological abnormalities seen in congenital muscle disorders and the potential for fixation to interfere with the recognition of these features, pathological evaluation of frozen muscle is preferable to fixed muscle when evaluating skeletal muscle for congenital muscle disease. Additionally, the potential to produce severe freezing artifacts in muscle requires specific precautions when freezing skeletal muscle for histological examination that are not commonly used when freezing other tissues. This manuscript describes a protocol for rapid freezing of skeletal muscle using isopentane (2-methylbutane) cooled with liquid nitrogen to preserve optimal skeletal muscle morphology. This procedure is also effective for freezing tissue intended for genetic or protein expression studies. Furthermore, we have integrated our freezing protocol into a broader procedure that also describes preferred methods for the short term triage of tissue for (1) single fiber functional studies and (2) myoblast cell culture, with a focus on the minimum effort necessary to collect tissue and transport it to specialized research or reference labs to complete these studies. Overall, this manuscript provides an outline of how fresh tissue can be effectively distributed for a variety of phenotypic studies and thereby provides standard operating procedures (SOPs) for pathological studies related to congenital muscle disease.

The analysis of skeletal muscle tissues to determine structural, functional, and biochemical properties is greatly facilitated by appropriate preparation. This protocol describes appropriate methods to prepare skeletal muscle tissue for a broad range of phenotyping studies.

Triage de tejidos y de congelación para los modelos de la enfermedad del músculo esquelético

Skeletal muscle is a unique tissue because of its structure and function, which requires specific protocols for tissue collection to obtain optimal ...