Ce travail a été soutenu par le NIDDK KO1 DK075386 subvention et l&#39;American Cancer Society RSG-09-075-01-MBC à juin dim.

Ce protocole comprend trois parties: la détection de culture bactérienne, gavage chez la souris, et la salmonelle. 1. Condition de la croissance de Salmonella. <ol><li> Préparer la Salmonella bouillon Luria-Bertani (LB) plaque, incuber à 37 ° C pendant la nuit. </li><li> Choisissez un clone de la plaque de LB et mis en 7 LB ml dans un tube de 12 ml et agiter à 37 ° C pendant environ 5 heures. </li><li> Inoculer 50 ml LB avec 0,05 ml d&#39;une culture en phase stationnaire et incuber à 37 ° C sans agitation pendant environ 18 heures. </li><li> Spin nuit culture bactérienne à la température ambiante à 6000 rpm pendant 10 minutes, de suspendre les bactéries dans HBSS utilisant 100:3 ratio (LB: HBSS). Par exemple: Chaque culture LB 50 ml sera suspendu dans 1,5 ml de HBSS. </li><li> Pour le gavage des animaux, diluer la culture bactérienne au ratio de 01:10. Par exemple: Chaque culture de 1,5 ml LB sera suspendu dans 15 ml de HBSS. </li></ol> 2. Salmonella-infection modèle *. <ol><li> Préparer la solution de streptomycine. Chaque souris sera donné 7,5 mg de streptomycine dans 100 ul de HBSS. Par exemple, préparer au total 90 mg de streptomycine dans 1,2 ml de HBSS pour les 10 souris. Toujours avoir un peu de volume supplémentaire lors de la préparation de la solution de streptomycine. </li><li> Retirer de l&#39;eau et de nourriture 4 heures avant le traitement par gavage oral. </li><li> Gavage des souris avec de la streptomycine avec 7,5 mg de streptomycine (100 pi de HBSS pour les souris témoins). Prenez la peau sur l&#39;épaule de la souris fermement avec le pouce et le majeur, étirer la tête et du cou avec l&#39;index pour faire l&#39;œsophage droite. Diriger la balle à pointe de l&#39;aiguille d&#39;alimentation sur le toit de la bouche et vers le côté droit du dos du pharynx, puis doucement descendre dans l&#39;œsophage et injecter la solution 100 ul. Aucune résistance devrait se faire sentir. </li><li> À 20 heures après le traitement à la streptomycine, l&#39;eau prélevée et de la nourriture à nouveau avant que les souris sont infectées par des bactéries. </li><li> Gavage chaque souris avec 100 ul de suspension dans HBSS ou traitées avec du HBSS stérile (contrôle) par gavage oral. La procédure de gavage est le même que 2,3) gavage des souris avec de la streptomycine. </li></ol> Les expérimentations animales ont été réalisées par * à l&#39;aide pathogènes spécifiques sans C57BL / 6 femelles souris (Taconic, Hudson, NY), qui étaient vieilles de 6-7 semaines, comme décrit précédemment 1. Le protocole a été approuvé par l&#39;Université de Rochester University Comité des Ressources Animales (UCAR). 3. Détection des salmonelles dans l&#39;intestin. <ol><li> Recueillir la souris fécales (environ 100 mg). </li><li> Transférer l&#39;échantillon fécal à un tube de 1,5 centrifugeuse micro avec 1ml de PBS et vortexer vigoureusement. </li><li> Centrifuger pendant 10 min à 800 rpm. Transférer le surnageant dans un tube de centrifugeuse propre. </li><li> Centrifugation à 6000 rpm pendant 5 min. Surnageant est jeté et 200 ul de PBS est ajouté aux pastilles et vortexés. </li><li> Streak le PBS 200 pi avec un épandeur portable jetable sur une plaque BBL CHROMagar pour détecter la bactérie Salmonella. Salmonella apparaissent mauves (rose au violet, voir Fig.1). Si 200 ul rendements trop nombreuses colonies sur la plaque, utiliser 100 ul ou 50 ul de la série. </li></ol> 4. Des résultats représentatifs. Lorsque le protocole est fait correctement, Salmonella typhimurium colonisation ne peut être détecté dans l&#39;intestin de la souris sur 6 mois. Salmonella pourrait être détectée par la culture fécale plus de 6 mois (Fig.1). Comme une des caractéristiques viennent de ce modèle, le corps la perte de poids et les décès surviennent dans les 4 semaines après l&#39;infection. Dépendant du souches de Salmonella utilisés pour l&#39;infection, certaines souris peuvent pas survivre plus de 6 mois. <img src="/files/ftp_upload/1947/1947fig1.jpg" alt="Figure 1" /> Figure 1. Salmonella intestinales chez les espèces de Salmonella apparaissent mauves (rose au violet) en couleur, en raison de différences métaboliques, en présence de chromogènes sélectionné. D&#39;autres bactéries sont soit inhibé ou produire des colonies bleu-vert ou incolore. <img src="/files/ftp_upload/1947/1947fig2.jpg" alt="Figure 2" /> Figure 2. Proportions de survie de souris infectées par la bactérie souche mutante PhoP c, c PhoP Avra-et PhoP c AvrA-/AvrA +, c PhoP Avra pendant 4 semaines (28 jours). Le poids corporel Tableau 1. De souris infectées par des salmonelles pendant 4 semaines. <table border="1"><tbody><tr><td></td><td> 0 </td><td> 1 semaine </td><td> 2 semaines </td><td> 3 semaines </td><td> 4 semaines </td></tr><tr><td> le contrôle </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> * Par rapport au groupe contrôle p &lt;0,05 ** Par rapport au groupe témoin p &lt;0,01 Tableau 2. Souches de Salmonella utilisé dans cette étude. <table border="1"><tbody><tr><td> Nom </td><td> Descriptif </td><td> Référence ou source </td></tr><tr><td> Salmonella 14028s </td><td> Wild-type S. typhimurium </td><td> ATCC </td></tr><tr><td> PhoP c </td><td> Non-pathogènes régulateur complexe mutant </td><td> Miller et al., 1990 </td></tr><tr><td> PhoP c Avra- </td><td> Avra-mutation </td><td> Collier-Hyams et al., 2002 </td></tr><tr><td> PhoP c AvrA-/AvrA + </td><td> PhoP c Avra-avec Avra encodage complété plasmidique </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>

Pour utiliser ce système, il est nécessaire pour le chercheur d&#39;apprendre à gavage des animaux. Nous détaillons une méthode pour préparer une culture bactérienne et gavage des souris. Nous montrons aussi comment contrôler la persistance de Salmonella dans le tractus gastro-intestinal (GI). Les étapes essentielles de ce protocole, y compris: <ol><li> Streptomycine prétraitement: streptomycine prétraitement pourrait se débarrasser de certains flore intestinale commensale et de faire des souris...

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chronic salmonella infected mouse model

Le modèle de souris infectées par des bactéries est un système puissant modèle pour étudier des domaines tels que l&#39;infection, l&#39;inflammation, l&#39;immunologie, la transduction du signal, et la tumorigenèse. De nombreux chercheurs ont tiré parti de la colite induite par<em&gt; Salmonella</emTyphimurium&gt; pour les études sur la phase précoce de l&#39;inflammation et l&#39;infection. Cependant, seuls quelques rapports sont sur l&#39;infection chronique<em&gt; In vivo</em&gt;. Souris avec<em&gt; Salmonella</emL&#39;existence&gt; persistante dans le tractus gastro-intestinal nous permettent d&#39;explorer la relation hôte-bactérienne à long terme d&#39;interaction, la transduction du signal, et la tumorigenèse. Nous avons établi un modèle de chroniques bactériennes de souris infectées par<em&gt; Salmonella</emLa colonisation typhimurium&gt; dans l&#39;intestin de la souris sur 6 mois. Pour utiliser ce système, il est nécessaire pour le chercheur d&#39;apprendre à préparer la culture bactérienne et gavage des animaux. Nous détaillons une méthode pour préparer des cultures bactériennes et des souris par gavage. Nous montrons aussi comment détecter la<em&gt; Salmonella</emLa persistance&gt; dans le tractus gastro-intestinal. Globalement, ce protocole aidera les chercheurs utilisant le modèle de souris infectées par des bactéries pour traiter des questions d&#39;importance fondamentale biologiques et microbiologiques.

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

Chronic Salmonella Infected Mouse Model

Établir un modèle de chroniques bactériennes de souris infectées par la persistance<em&gt; Salmonella</emLa colonisation typhimurium&gt; dans l&#39;intestin pendant 27 semaines.

Chronique Salmonella Modèle de souris infectées

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 vues.  University of Rochester. Établir un modèle de chroniques bactériennes de souris infectées par la persistance Salmonella dans l'intestin pendant 27 semaines.

Vidéo: 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.

Modèle de souris orthotopique du cancer colorectal

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

Recherche

Biologie

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.

Laser-Induced modèle chronique hypertension oculaire sur des rats 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.

L&#39;immunohistochimie sur coupes en paraffine d&#39;épiderme de souris en utilisant des anticorps fluorescents

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.

L&#39;imagerie in vivo et des traitements thérapeutiques dans un modèle de souris orthotopique de cancer des ovaires

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.

Génération de souris dérivées de cellules souches pluripotentes induites

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

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.

Le murin déficients en Choline, additionné d’éthionine (CDE) alimentation modèle d’atteinte hépatique chronique

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-intérieur cellulaire massive Injection de cellules souches embryonnaires conduit à des taux plus élevés de chimérisme

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

A Mouse Model of Intestinal Partial Obstruction

Intestinal obstructions, that impede or block peristaltic movement, can be caused by abdominal adhesions and most gastrointestinal (GI) diseases including tumorous growths. However, the cellular remodeling mechanisms involved in, and caused by, intestinal obstructions are poorly understood. Several animal models of intestinal obstructions have been developed, but the mouse model is the most cost/time effective. The mouse model uses the surgical implantation of an intestinal partial obstruction (PO) that has a high mortality rate if it is not performed correctly. In addition, mice receiving PO surgery fail to develop hypertrophy if an appropriate blockade is not used or not properly placed. Here, we describe a detailed protocol for PO surgery which produces reliable and reproducible intestinal obstructions with a very low mortality rate. This protocol utilizes a surgically placed silicone ring that surrounds the ileum which partially blocks digestive movement in the small intestine. The partial blockage makes the intestine become dilated due to the halt of digestive movement. The dilation of the intestine induces smooth muscle hypertrophy on the oral side of the ring that progressively develops over 2 weeks until it causes death. The surgical PO mouse model offers an in vivo model of hypertrophic intestinal tissue useful for studying pathological changes of intestinal cells including smooth muscle cells (SMC), interstitial cells of Cajal (ICC), PDGFR&#945;+, and neuronal cells during the development of intestinal obstruction.

Intestinal obstructions are a partial or complete blockage of the intestine that can cause severe abdominal pain, nausea, vomiting, and preventing the passage of stool. This procedure for creating intestinal partial obsructions in mice is reliable in studying the mechanisms underlying pathological cell growth and death in the intestine.

Un modèle de souris d’une Obstruction intestinale partielle

Intestinal obstructions, that impede or block peristaltic movement, can be caused by abdominal adhesions and most gastrointestinal (GI) diseases including ...

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;

Quasi-métagénomique analyse des Salmonella dans les aliments et les échantillons environnementaux

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.