Este trabajo fue apoyado por el NIDDK KO1 DK075386 subvención y la Sociedad Americana del Cáncer RSG-09-075-01-MBC de junio dom

Este protocolo incluye tres partes: la detección de un cultivo bacteriano, una sonda de ratón, y Salmonella. 1. Salmonella condiciones de crecimiento. <ol><li> Prepare la Salmonella caldo Luria-Bertani (LB) placa, se incuba a 37 ° C durante la noche. </li><li> Elige un clon de la placa de LB y puesto en LB 7 ml en un tubo de 12 ml y agitar a 37 º C durante 5 horas. </li><li> Inocular 50 ml de LB con 0,05 ml de un cultivo en fase estacionaria y se incuba a 37 ° C sin agitación durante aproximadamente 18 horas. </li><li> Giro cultivo bacteriano durante la noche a temperatura ambiente con 6000 rpm durante 10 minutos, suspender las bacterias en HBSS con relación 100:3 (LB: HBSS). Por ejemplo: Cada cultura LB 50 ml se suspenderá en 1,5 ml HBSS. </li><li> Para la alimentación forzada de animales, diluir aún más el cultivo bacteriano en una proporción de 1:10. Por ejemplo: Todas las culturas de 1,5 ml LB será suspendido en 15 ml HBSS. </li></ol> 2. Salmonella infección modelo *. <ol><li> Prepare la solución de estreptomicina. Cada ratón se le dará 7,5 mg de estreptomicina en 100 l HBSS. Por ejemplo, prepare un total de 90 mg de estreptomicina en 1,2 ml de HBSS 10 ratones. Siempre tienen algo de volumen extra en la preparación de la solución de estreptomicina. </li><li> Retirar el agua y la comida 4 horas antes del tratamiento de una sonda nasogástrica. </li><li> Ratones sonda con estreptomicina con 7,5 mg de estreptomicina (100 l de HBSS para los ratones de control). Coge la piel sobre el hombro del ratón firmemente con el pulgar y el dedo medio, estirar la cabeza y el cuello con el dedo índice para hacer que el esófago directamente. Dirigir el balón de punta de la aguja de alimentación a lo largo del techo de la boca y hacia el lado derecho de la parte posterior de la faringe, y luego suavemente pase hacia el esófago y se inyecta la solución de 100 l. No hay resistencia se debe sentir. </li><li> A las 20 horas después del tratamiento con estreptomicina, el agua extraída y la comida de nuevo antes de los ratones infectados con la bacteria. </li><li> Sonda cada ratón con 100 l de suspensión en HBSS o tratados con HBSS estéril (control) mediante una sonda nasogástrica. El procedimiento de alimentación forzada es lo mismo que 2.3) ratones sonda con estreptomicina. </li></ol> * Experimentos con animales se realizaron mediante el uso de patógenos específicos libre C57BL mujeres / 6 ratones (Taconic, Hudson, Nueva York), que fueron 6-7 semanas de edad, como se describió anteriormente 1. El protocolo fue aprobado por la Universidad de la Universidad de Rochester Comité de Recursos Animales (UCAR). 3. La detección de Salmonella en el intestino. <ol><li> Recoger las heces de ratón (100 mg). </li><li> Transferencia de la muestra fecal en un tubo de centrífuga de 1,5 micro con 1 ml de PBS y agitar vigorosamente. </li><li> Centrifugar durante 10 minutos a 800 rpm. Transferir el sobrenadante a un tubo de microcentrífuga limpio. </li><li> Centrifugación a 6000 rpm durante 5 minutos. Sobrenadante se descarta y 200 l de PBS se añade a los gránulos y vortex. </li><li> La racha de 200 l de PBS con un esparcidor celular desechable en una placa BBL CHROMagar para detectar Salmonella. Especies de Salmonella aparecen de color malva (rosa a púrpura, ver figura 1). Si los rendimientos de 200 l colonias demasiados en la placa, utilice 100 l ó 50 l durante la racha. </li></ol> 4. Los resultados representativos. Cuando el protocolo se hace correctamente, Salmonella typhimurium colonización se puede detectar en el intestino del ratón más de 6 meses. Salmonella pueden ser detectados por cultivo fecal más de 6 meses (Fig. 1). Como fuera típica vienen de este modelo, el cuerpo de pérdida de peso y la muerte ocurren dentro de las 4 semanas después de la infección. Depende de las cepas de Salmonella utilizados para la infección, algunos ratones no pueden sobrevivir más de 6 meses. <img src="/files/ftp_upload/1947/1947fig1.jpg" alt="Figura 1" /> Figura 1. Salmonella Intestinal en la especie Salmonella aparecen de color malva (rosa a púrpura) en color, debido a las diferencias metabólicas en presencia de cromógenos seleccionado. Otras bacterias son inhibidas o producen colonias de color azul-verdoso o incoloro. <img src="/files/ftp_upload/1947/1947fig2.jpg" alt="Figura 2" /> Figura 2. Proporciones de supervivencia de ratones infectados con Salmonella cepa mutante c PhoP, PhoP c Avra, y PhoP c AvrA-/AvrA +, c PhoP Avra durante 4 semanas (28 días). Tabla 1. Peso corporal de los ratones infectados con Salmonella durante 4 semanas. <table border="1"><tbody><tr><td></td><td> 0 </td><td> 1 semana </td><td> 2 semanas </td><td> 3 semanas </td><td> 4 semanas </td></tr><tr><td> control </td><td> 16,78 ± 1,05 </td><td> 16,91 ± 1,28 </td><td> 18,26 ± 1,23 </td><td> 19,31 ± 1,26 </td><td> 20,26 ± 1,15 </td></tr><tr><td> PhoP c </td&gt; <td> 16,89 ± 1,03 </td><td> 17,14 ± 1,19 </td><td> 17,43 ± 1,63 * </td><td> 18,68 ± 1,78 </td><td> 20,05 ± 1,11 </td></tr><tr><td> PhoP c Avra- </td><td> 16,91 ± 1,12 </td><td> 16,96 ± 1,39 </td><td> 17,06 ± 2,14 ** </td><td> 18,71 ± 2,18 </td><td> 20,15 ± 1,56 </td></tr><tr><td> PhoP c AvrA-/AvrA + </td><td> 16,94 ± 0,96 </td><td> 17,17 ± 1,02 </td><td> 17,63 ± 1,42 * </td><td> 18,44 ± 2,03 </td><td> 20,09 ± 1,17 </td></tr></tbody></table> * En comparación con el grupo control p &lt;0,05 ** En comparación con el grupo control p &lt;0,01 Tabla 2. Cepas de Salmonella utilizada en este estudio. <table border="1"><tbody><tr><td> Nombre </td><td> Descripción </td><td> Referencia o fuente </td></tr><tr><td> Salmonella 14028s </td><td> De tipo salvaje S. typhimurium </td><td> ATCC </td></tr><tr><td> PhoP c </td><td> No patógenos mutantes regulador complejo </td><td> Miller et al., 1990 </td></tr><tr><td> PhoP c Avra- </td><td> Avra-mutación </td><td> Collier-Hyams et al., 2002 </td></tr><tr><td> PhoP c AvrA-/AvrA + </td><td> PhoP c Avra Avra complementa con plásmido que codifica </td><td> Collier-Hyams et al., 2002 </td></tr></tbody></table>

<ol>
	<li>Duan, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=beta-Catenin+activity+negatively+regulates+bacteria-induced+inflammation.">beta-Catenin activity negatively regulates bacteria-induced inflammation.</a> Lab Invest. 87 (6), 613-624 (2007).</li><li>Barthel, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pretreatment+of+mice+with+streptomycin+provides+a+Salmonella+enterica+serovar+Typhimurium+colitis+model+that+allows+analysis+of+both+pathogen+and+host.">Pretreatment of mice with streptomycin provides a Salmonella enterica serovar Typhimurium colitis model that allows analysis of both pathogen and host.</a> Infection and immunity. 71 (5), 2839-2858 (2003).</li><li>Miller, S. I., Mekalanos, 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=Constitutive+expression+of+the+phoP+regulon+attenuates+Salmonella+virulence+and+survival+within+macrophages.">Constitutive expression of the phoP regulon attenuates Salmonella virulence and survival within macrophages.</a> Journal of bacteriology. 172 (5), 2485-2490 (1990).</li><li>Valdez, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nramp1+expression+by+dendritic+cells+modulates+inflammatory+responses+during+Salmonella+Typhimurium+infection.">Nramp1 expression by dendritic cells modulates inflammatory responses during Salmonella Typhimurium infection.</a> Cell Microbiol. 10 (8), 1646-1661 (2008).</li><li>Woo, H., Okamoto, S., Guiney, D., Gunn, J. S., Fierer, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+model+of+Salmonella+colitis+with+features+of+diarrhea+in+SLC11A1+wild-type+mice.">A model of Salmonella colitis with features of diarrhea in SLC11A1 wild-type mice.</a> PLoS ONE. 3 (2), e1603-e1603 (2008).</li><li>Dangl, 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=Molecular+call-and-response:+how+Salmonella+learns+the+gospel+from+its+host.">Molecular call-and-response: how Salmonella learns the gospel from its host.</a> Trends Microbiol. 11 (6), 245-246 (2003).</li><li>Zaharik, M. L., Vallance, B. A., Puente, J. L., Gros, P., Finlay, B. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Host-pathogen+interactions:+Host+resistance+factor+Nramp1+up-regulates+the+expression+of+Salmonella+pathogenicity+island-2+virulence+genes.">Host-pathogen interactions: Host resistance factor Nramp1 up-regulates the expression of Salmonella pathogenicity island-2 virulence genes.</a> Proceedings of the National Academy of Sciences of the United States of America. 99, 15705-15710 (2002).</li><li>Townsend, P., Morton, 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=Laboratory+Animal+Care+Policies+and+Regulations:+United+Kingdom.">Laboratory Animal Care Policies and Regulations: United Kingdom.</a> ILAR J. 37 (2), 68-74 (1995).</li><li>Olfert, E. D., Godson, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Humane+endpoints+for+infectious+disease+animal+models.">Humane endpoints for infectious disease animal models.</a> ILAR J. 41 (2), 99-104 (2000).</li><li>Ye, Z., Petrof, E. O., Boone, D., Claud, E. C., Sun, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Salmonella+effector+AvrA+regulation+of+colonic+epithelial+cell+inflammation+by+deubiquitination.">Salmonella effector AvrA regulation of colonic epithelial cell inflammation by deubiquitination.</a> Am J Pathol. 171 (3), 882-892 (2007).</li><li>Grassl, G. A., Valdez, Y., Bergstrom, K. S., Vallance, B. A., Finlay, B. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chronic+enteric+salmonella+infection+in+mice+leads+to+severe+and+persiste+nt+intestinal+fibrosis.">Chronic enteric salmonella infection in mice leads to severe and persiste nt intestinal fibrosis.</a> Gastroenterology. 134 (3), 768-780 (2008).</li><li>Sukupolvi, S., Edelstein, A., Rhen, M., Normark, S. J., Pfeifer, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+a+murine+model+of+chronic+Salmonella+infection.">Development of a murine model of chronic Salmonella infection.</a> Infection and immunity. 65 (2), 838-842 (1997).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Biosafety in Microbiological and Biomedical Laboratories (BMBL). , (2007).</li><li>Wu, S., Lu, R., Zhang, Y., Sun, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chronic+effects+of+a+Salmonella+type+III+secretion+effector+protein+AvrA+in+vivo.">Chronic effects of a Salmonella type III secretion effector protein AvrA in vivo.</a> PLoS ONE. , (2010).</li></ol>

Para utilizar este sistema, es necesario que el investigador que aprender a alimentación forzada de los animales. Se detalla una metodología para preparar un cultivo bacteriano y alimentación forzada de los ratones. También muestra cómo controlar la persistencia de Salmonella en el tracto gastrointestinal (GI). Los pasos críticos de este protocolo, incluyendo: <ol><li> Estreptomicina pretratamiento: estreptomicina pretratamiento podría deshacerse de algunos flora intestinal comensal y que los ratones...

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<td>HBSS</td>
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<td>Sigma</td>
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chronic salmonella infected mouse model

El modelo de ratón infectado de bacterias es un sistema poderoso modelo para el estudio de áreas tales como infección, inflamación, la inmunología, la transducción de señales y tumorigénesis. Muchos investigadores han tomado ventaja de la colitis inducida por<em&gt; Salmonella</em&gt; Typhimurium para los estudios en la fase temprana de la inflamación y la infección. Sin embargo, sólo unos pocos informes se encuentran en la infección crónica<em&gt; In vivo</em&gt;. Los ratones con<em&gt; Salmonella</emExistencia&gt; persistente en el tracto gastrointestinal nos permiten explorar el anfitrión-bacterial interacción a largo plazo, la transducción de señales y tumorigénesis. Hemos establecido un modelo de crónica bacteriana ratones infectados con el<em&gt; Salmonella</em&gt; Typhimurium colonización en el intestino del ratón más de 6 meses. Para utilizar este sistema, es necesario que el investigador para aprender a preparar el cultivo de bacterias y alimentación forzada de los animales. Se detalla una metodología para el cultivo de bacterias y preparar a los ratones por sonda nasogástrica. También muestra la forma de detectar la<em&gt; Salmonella</em&gt; Persistencia en el tracto gastrointestinal. En términos generales, este protocolo ayudará a los investigadores con el modelo de ratón infectado de bacterias para hacer frente a cuestiones biológicas y microbiológicas de fundamental importancia.

Watch this Scientific Journal Video about Chronic Salmonella Infected Mouse Model at JoVE.com

Chronic Salmonella Infected Mouse Model

Establecer un modelo de crónica bacteriana ratones infectados con el persistente<em&gt; Salmonella</em&gt; Typhimurium colonización en el intestino durante 27 semanas.

Crónico Salmonella Modelo de ratón infectado

The bacterial infected mouse model is a powerful model system for studying areas such as infection, inflammation, immunology, signal transduction, and ...

Research

JoVE Journal

Biology

Chronic Salmonella Infected Mouse Model

17.2K vistas.  University of Rochester. Establecer un modelo de crónica bacteriana ratones infectados con el persistente Salmonella Typhimurium colonización en el intestino durante 27 semanas.

Video: Chronic Salmonella Infected Mouse Model

Orthotopic Mouse Model of Colorectal Cancer

The traditional subcutaneous tumor model is less than ideal for studying colorectal cancer. Orthotopic mouse models of colorectal cancer, which feature cancer cells growing in their natural location, replicate human disease with high fidelity. Two techniques can be used to establish this model. Both techniques are similar and require mouse anesthesia and laparotomy for exposure of the cecum. One technique involves injection of a colorectal cancer cell suspension into the cecal wall. Cancer cells are first grown in culture, harvested when subconfluent and prepared as a single cell suspension. A small volume of cells is injected slowly to avoid leakage. The other technique involves transplantation of a piece of subcutaneous tumor onto the cecum. A mouse with a previously established subcutaneous colorectal tumor is euthanized and the tumor is removed using sterile technique. The tumor piece is divided into small pieces for transplantation to another mouse. Prior to transplantation, the cecal wall is lightly damaged to facilitate tumor cell infiltration. The time to developing primary tumors and liver metastases will vary depending on the technique, cell line, and mouse species used. This orthotopic mouse model is useful for studying the natural progression of colorectal cancer and testing new therapeutic agents against colorectal cancer.

Two techniques can be used to establish this model: injection of a cancer cell suspension into the cecal wall or transplantation of a piece of subcutaneous tumor onto the cecum. This model is useful for studying the natural progression of colorectal cancer and testing new therapeutic agents against colorectal cancer.

Modelo de ratón ortotópico de cáncer colorrectal

The traditional subcutaneous tumor model is less than ideal for studying colorectal cancer. Orthotopic mouse models of colorectal cancer, which feature ...

Investigación

Biología

Laser-Induced Chronic Ocular Hypertension Model on SD Rats

Glaucoma is one of the major causes of blindness in the world. Elevated intraocular pressure is a major risk factor. Laser photocoagulation induced ocular hypertension is one of the well established animal models. This video demonstrates how to induce ocular hypertension by Argon laser photocoagulation in rat.

Glaucoma is one of the major causes of blindness in the world. Elevated intraocular pressure is a major risk factor. Laser photocoagulation induced ocular hypertension is one of the well established animal models. This video demonstrates how to induce ocular hypertension by Argon laser photocoagulation in rat.

Inducida por láser ocular crónica modelo de hipertensión en ratas SD

Glaucoma is one of the major causes of blindness in the world. Elevated intraocular pressure is a major risk factor. Laser photocoagulation induced ocular ...

Immunohistochemistry on Paraffin Sections of Mouse Epidermis Using Fluorescent Antibodies

In the epidermis, immunohistochemistry is an efficient means of localizing specific proteins to their relative expression compartment; namely the basal, suprabasal, and stratum corneum layers. The precise localization within the epidermis of a particular protein lends clues toward its functional role within the epidermis. In this chapter, we describe a reliable method for immunolocalization within the epidermis modified for both frozen and paraffin sections that we use very routinely in our laboratory. Paraffin sections generally provide much better morphology, hence, superior results and photographs; however, not all antibodies will work with the harsh fixation and treatment involved in their processing. Therefore, the protocol for frozen sectioning is also included. Within paraffin sectioning, two fixation protocols are described (Bouin's and paraformaldehyde); the choice of fixative will be directly related to the antibody specifications and may require another fixing method.

We describe a reliable method for immunolocalization within the epidermis modified for both frozen and paraffin sections that we use very routinely in our laboratory.

Inmunohistoquímica en secciones de parafina de la epidermis del ratón Utilizando anticuerpos fluorescentes

In the epidermis, immunohistochemistry is an efficient means of localizing specific proteins to their relative expression compartment; namely the basal, ...

In vivo Imaging and Therapeutic Treatments in an Orthotopic Mouse Model of Ovarian Cancer

Human cancer and response to therapy is better represented in orthotopic animal models. This paper describes the development of an orthotopic mouse model of ovarian cancer, treatment of cancer via oral delivery of drugs, and monitoring of tumor cell behavior in response to drug treatment in real time using in vivo imaging system. In this orthotopic model, ovarian tumor cells expressing luciferase are applied topically by injecting them directly into the mouse bursa where each ovary is enclosed. Upon injection of D-luciferin, a substrate of firefly luciferase, luciferase-expressing cells generate bioluminescence signals. This signal is detected by the in vivo imaging system and allows for a non-invasive means of monitoring tumor growth, distribution, and regression in individual animals. Drug administration via oral gavage allows for a maximum dosing volume of 10 mL/kg body weight to be delivered directly to the stomach and closely resembles delivery of drugs in clinical treatments. Therefore, techniques described here, development of an orthotopic mouse model of ovarian cancer, oral delivery of drugs, and in vivo imaging, are useful for better understanding of human ovarian cancer and treatment and will improve targeting this disease.

In vivo Imaging and Therapeutic Treatments in an Orthotopic Mouse Model of Ovarian Cancer

Orthotopic animal models of ovarian cancer replicate better human disease and therefore enhance our understanding of cancer progression and tumor response to therapy. A mouse model receives an intrabursal injection of luciferase-expressing ovarian tumor cells. Treatment is administered via oral gavage. Tumor growth is monitored by in vivo imaging system.

En vivo de imágenes y tratamientos terapéuticos en un modelo de ratón ortotópico de cáncer de ovario

Human cancer and response to therapy is better represented in orthotopic animal models.  This paper describes the development of an orthotopic mouse model ...

Generation of Mice Derived from Induced Pluripotent Stem Cells

The production of induced pluripotent stem cells (iPSCs) from somatic cells provides a means to create valuable tools for basic research and may also produce a source of patient-matched cells for regenerative therapies. iPSCs may be generated using multiple protocols and derived from multiple cell sources. Once generated, iPSCs are tested using a variety of assays including immunostaining for pluripotency markers, generation of three germ layers in embryoid bodies and teratomas, comparisons of gene expression with embryonic stem cells (ESCs) and production of chimeric mice with or without germline contribution2. Importantly, iPSC lines that pass these tests still vary in their capacity to produce different differentiated cell types2. This has made it difficult to establish which iPSC derivation protocols, donor cell sources or selection methods are most useful for different applications.
The most stringent test of whether a stem cell line has sufficient developmental potential to generate all tissues required for survival of an organism (termed full pluripotency) is tetraploid embryo complementation (TEC)3-5. Technically, TEC involves electrofusion of two-cell embryos to generate tetraploid (4n) one-cell embryos that can be cultured in vitro to the blastocyst stage6. Diploid (2n) pluripotent stem cells (e.g. ESCs or iPSCs) are then injected into the blastocoel cavity of the tetraploid blastocyst and transferred to a recipient female for gestation (see Figure 1). The tetraploid component of the complemented embryo contributes almost exclusively to the extraembryonic tissues (placenta, yolk sac), whereas the diploid cells constitute the embryo proper, resulting in a fetus derived entirely from the injected stem cell line.
Recently, we reported the derivation of iPSC lines that reproducibly generate adult mice via TEC1. These iPSC lines give rise to viable pups with efficiencies of 5-13%, which is comparable to ESCs3,4,7 and higher than that reported for most other iPSC lines8-12. These reports show that direct reprogramming can produce fully pluripotent iPSCs that match ESCs in their developmental potential and efficiency of generating pups in TEC tests. At present, it is not clear what distinguishes between fully pluripotent iPSCs and less potent lines13-15. Nor is it clear which reprogramming methods will produce these lines with the highest efficiency. Here we describe one method that produces fully pluripotent iPSCs and "all- iPSC" mice, which may be helpful for investigators wishing to compare the pluripotency of iPSC lines or establish the equivalence of different reprogramming methods.

Generating induced pluripotent stem cell (iPSC) lines produces lines of differing developmental potential even when they pass standard tests for pluripotency. Here we describe a protocol to produce mice derived entirely from iPSCs, which defines the iPSC lines as possessing full pluripotency1.

Generación de ratones derivados de células madre pluripotentes inducidas

The production of induced pluripotent stem cells (iPSCs) from somatic cells provides a means to create valuable tools for basic research and may also ...

Maintaining Aedes aegypti Mosquitoes Infected with Wolbachia

Aedes aegypti mosquitoes experimentally infected with Wolbachia are being utilized in programs to control the spread of arboviruses such as dengue, chikungunya and Zika. Wolbachia-infected mosquitoes can be released into the field to either reduce population sizes through incompatible matings or to transform populations with mosquitoes that are refractory to virus transmission. For these strategies to succeed, the mosquitoes released into the field from the laboratory must be competitive with native mosquitoes. However, maintaining mosquitoes in the laboratory can result in inbreeding, genetic drift and laboratory adaptation which can reduce their fitness in the field and may confound the results of experiments. To test the suitability of different Wolbachia infections for deployment in the field, it is necessary to maintain mosquitoes in a controlled laboratory environment across multiple generations. We describe a simple protocol for maintaining Ae. aegypti mosquitoes in the laboratory, which is suitable for both Wolbachia-infected and wild-type mosquitoes. The methods minimize laboratory adaptation and implement outcrossing to increase the relevance of experiments to field mosquitoes. Additionally, colonies are maintained under optimal conditions to maximize their fitness for open field releases.

Maintaining Aedes aegypti Mosquitoes Infected with Wolbachia

Aedes aegypti mosquitoes infected with Wolbachia are being released into natural populations to suppress the transmission of arboviruses. We describe methods to rear Ae. aegypti with Wolbachia infections in the laboratory for experiments and field release, taking precautions to minimize laboratory adaptation and selection.

Mantener los mosquitos Aedes aegypti infectados con Wolbachia

Aedes aegypti mosquitoes experimentally infected with Wolbachia are being utilized in programs to control the spread of arboviruses such as dengue, ...

The Murine Choline-Deficient, Ethionine-Supplemented (CDE) Diet Model of Chronic Liver Injury

Chronic liver diseases, such as viral hepatitis, alcoholic liver disease, or non-alcoholic fatty liver disease, are characterized by continual inflammation, progressive destruction and regeneration of the hepatic parenchyma, liver progenitor cell proliferation, and fibrosis. The end-stage of every chronic liver disease is cirrhosis, a major risk factor for the development of hepatocellular carcinoma. To study processes regulating disease initiation, establishment, and progression, several animal models are used in laboratories. Here we describe a six-week time course of the choline-deficient and ethionine-supplemented (CDE) mouse model, which involves feeding six-week old male C57BL/6J mice with choline-deficient chow and 0.15% DL-ethionine-supplemented drinking water. Monitoring of animal health and a typical body weight loss curve are explained. The protocol demonstrates the gross examination of a CDE-treated liver and blood collection by cardiac puncture for subsequent serum analyses. Next, the liver perfusion technique and collection of different hepatic lobes for standard evaluations are shown, including liver histology assessments by hematoxylin and eosin or Sirius Red stainings, immunofluorescent detection of hepatic cell populations as well as transcriptome profiling of the liver microenvironment. This mouse model is suitable for studying inflammatory, fibrogenic, and liver progenitor cell dynamics induced through chronic liver disease and can be used to test potential therapeutic agents that may modulate these processes.

The Murine Choline-Deficient, Ethionine-Supplemented CDE Diet Model of Chronic Liver Injury

Here we describe a common method to induce chronic liver injury in mice by feeding of a choline-deficient and ethionine-supplemented (CDE) diet. We demonstrate health monitoring, liver perfusion, isolation, and preservation. A time course of six weeks can inform about liver injury, pathohistology, fibrosis, inflammatory, and liver progenitor cell responses.

El murino colina deficientes, suplementada con Ethionine (CDE) dieta de modelo de la lesión hepática crónica

Chronic liver diseases, such as viral hepatitis, alcoholic liver disease, or non-alcoholic fatty liver disease, are characterized by continual ...

Trans-inner Cell Mass Injection of Embryonic Stem Cells Leads to Higher Chimerism Rates

In an effort to increase efficiency in the creation of genetically modified mice via ES Cell methodologies, we present an adaptation to the current blastocyst injection protocol. Here we report that a simple rotation of the embryo, and injection through Trans-Inner cell mass (TICM) increased the percentage of chimeric mice from 31% to 50%, with no additional equipment or further specialized training. 26 different inbred clones, and 35 total clones were injected over a period of 9 months. There was no significant difference in either pregnancy rate or recovery rate of embryos between traditional injection techniques and TICM. Therefore, without any major alteration in the injection process and a simple positioning of the blastocyst and injecting through the ICM, releasing the ES cells into the blastocoel cavity can potentially improve the quantity of chimeric production and subsequent germline transmission.

Here we present a protocol to increase chimera production without the use of new equipment. A simple orientation change of the embryo for injection can increase the number of embryos produced, and potentially reduce the timeline to germline transmission.

Trans-interno celular masiva inyección de células madre embrionarias conduce a mayores tasas de quimerismo

In an effort to increase efficiency in the creation of genetically modified mice via ES Cell methodologies, we present an adaptation to the current ...

Quasi-metagenomic Analysis of Salmonella from Food and Environmental Samples

Quasi-metagenomics sequencing refers to the sequencing-based analysis of modified microbiomes of food and environmental samples. In this protocol, microbiome modification is designed to concentrate genomic DNA of a target foodborne pathogen contaminant to facilitate the detection and subtyping of the pathogen in a single workflow. Here, we explain and demonstrate the sample preparation steps for the quasi-metagenomics analysis of Salmonella enterica from representative food and environmental samples including alfalfa sprouts, ground black pepper, ground beef, chicken breast and environmental swabs. Samples are first subjected to the culture enrichment of Salmonella for a shortened and adjustable duration (4&#8211;24 h). Salmonella cells are then selectively captured from the enrichment culture by immunomagnetic separation (IMS). Finally, multiple displacement amplification (MDA) is performed to amplify DNA from IMS-captured cells. The DNA output of this protocol can be sequenced by high throughput sequencing platforms. An optional quantitative PCR analysis can be performed to replace sequencing for Salmonella detection or assess the concentration of Salmonella DNA before sequencing.

Quasi-metagenomic Analysis of Salmonella from Food and Environmental Samples

Here, we present a protocol to prepare DNA samples from food and environmental microbiomes for concerted detection and subtyping of Salmonella through quasimetagenomic sequencing. The combined use of culture enrichment, immunomagnetic separation (IMS), and multiple displacement amplification (MDA) allows effective concentration of Salmonella genomic DNA from food and environmental samples.&#160;

Análisis de metagenómica cuasi de Salmonella de muestras alimentarias y ambientales

Quasi-metagenomics sequencing refers to the sequencing-based analysis of modified microbiomes of food and environmental samples. In this protocol, ...

Mass Spectrometry Analysis to Identify Ubiquitylation of EYFP-tagged CENP-A (EYFP-CENP-A)

Studying the structure and the dynamics of kinetochores and centromeres is important in understanding chromosomal instability (CIN) and cancer progression. How the chromosomal location and function of a centromere (i.e., centromere identity) are determined and participate in accurate chromosome segregation is a fundamental question. CENP-A is proposed to be the non-DNA indicator (epigenetic mark) of centromere identity, and CENP-A ubiquitylation is required for CENP-A deposition at the centromere, inherited through dimerization between cell division, and indispensable to cell viability.
Here we describe mass spectrometry analysis to identify ubiquitylation of EYFP-CENP-A K124R mutant suggesting that ubiquitylation at a different lysine is induced because of the EYFP tagging in the CENP-A K124R mutant protein. Lysine 306 (K306) ubiquitylation in EYFP-CENP-A K124R was successfully identified, which corresponds to lysine 56 (K56) in CENP-A through mass spectrometry analysis. A caveat is discussed in the use of GFP/EYFP or the tagging of high molecular weight protein as a tool to analyze the function of a protein. Current technical limit is also discussed for the detection of ubiquitylated bands, identification of site-specific ubiquitylation(s), and visualization of ubiquitylation in living cells or a specific single cell during the whole cell cycle.
The method of mass spectrometry analysis presented here can be applied to human CENP-A protein with different tags and other centromere-kinetochore proteins. These combinatory methods consisting of several assays/analyses could be recommended for researchers who are interested in identifying functional roles of ubiquitylation.

Mass Spectrometry Analysis to Identify Ubiquitylation of EYFP-tagged CENP-A EYFP-CENP-A

CENP-A ubiquitylation is an important requirement for CENP-A deposition at the centromere, inherited through dimerization between cell division, and indispensable to cell viability. Here we describe mass spectrometry analysis to identify ubiquitylation of EYFP-tagged CENP-A (EYFP-CENP-A) protein.