Este trabalho foi financiado pelo NIDDK KO1 DK075386 subvenção ea Sociedade Americana do Câncer RSG-09-075-01-MBC para junho dom

Este protocolo inclui três partes: detecção de cultura bacteriana, gavage mouse, e Salmonella. 1. Condições de crescimento Salmonella. <ol><li> Prepare a Salmonella Luria-Bertani caldo de placa (LB), incubar a 37 ° C durante a noite. </li><li> Escolha um clone da placa de LB e colocar em 7 LB ml em um tubo de 12 ml, e agitar a 37 ° C por cerca de 5 horas. </li><li> Inocular 50 ml LB com 0,05 ml de uma cultura de fase estacionária e incubar a 37 ° C sem agitação por cerca de 18 horas. </li><li> Rotação de cultura bacteriana durante a noite a temperatura ambiente com 6000 rpm por 10 minutos, suspender as bactérias em HBSS usando 100:3 ratio (LB: HBSS). Por exemplo: Cada cultura LB 50 ml será suspenso em 1,5 ml HBSS. </li><li> Para a sonda animal, diluir a cultura bacteriana na proporção de 1:10. Por exemplo: A cada 1,5 ml de cultura LB será suspenso em 15 ml HBSS. </li></ol> 2. Salmonella modelo de infecção *. <ol><li> Preparar a solução de estreptomicina. Cada rato será dado 7,5 mg de estreptomicina em 100 mL HBSS. Por exemplo, prepare mg 90 total de estreptomicina, em 1,2 ml de HBSS 10 ratos. Sempre tem algum volume extra na preparação da solução de estreptomicina. </li><li> Retirar água e comida 4 horas antes do tratamento gavagem oral. </li><li> Camundongos gavagem com estreptomicina com 7,5 mg de estreptomicina (100 l de HBSS para os ratos de controle). Agarre a pele sobre o ombro do mouse firmemente com o polegar eo dedo médio, esticar a cabeça e pescoço com o dedo indicador para fazer o esôfago em linha reta. Dirigir a bola ponta da agulha de alimentação ao longo do céu da boca e em direção ao lado direito da parte de trás da faringe, em seguida, delicadamente passar para dentro do esôfago e injetar a solução de 100 ml. Nenhuma resistência deve ser sentida. </li><li> Menos 20 horas após o tratamento estreptomicina, água e alimentos retirados novamente antes de os camundongos são infectados com bactérias. </li><li> Gavagem cada rato com 100 mL de suspensão em HBSS ou tratados com HBSS estéril (controle) por gavagem oral. O procedimento de gavagem é o mesmo que 2.3) ratos gavage com estreptomicina. </li></ol> Experimentos em animais * foram realizadas utilizando-específicas isentos de agentes patogénicos C57BL feminino / 6 camundongos (Taconic, Hudson, NY), que foram 6-7 semanas de idade, como descrito anteriormente 1. O protocolo foi aprovado pela Universidade de Rochester University Comité de Recursos Animal (UCAR). 3. Detecção de Salmonella no intestino. <ol><li> Coletar rato fecal (cerca de 100 mg). </li><li> Transferência da amostra fecal a um tubo de centrífuga de 1,5 micro com 1ml de PBS e vortex vigorosamente. </li><li> Centrifugar por 10 min a 800 rpm. Transferir o sobrenadante para um tubo de microcentrífuga limpo. </li><li> Centrifugação a 6000 rpm por 5 min. Sobrenadante é descartado e 200 mL PBS é adicionado à pellets e agitadas. </li><li> Raia o PBS 200 mL com um espalhador de células em um prato descartável CHROMagar BBL para detectar Salmonella. Espécies Salmonella aparecem malva (rosa ao roxo, ver Fig.1). Se 200 mL rende muitas colônias na placa, use 100 ml ou 50 ul para a raia. </li></ol> 4. Resultados representante. Quando o protocolo é feito corretamente, a colonização de Salmonella typhimurium pode ser detectado no intestino do rato mais de 6 meses. Salmonella pode ser detectado por cultura fecal mais de 6 meses (Fig.1). Como fora típico vêm deste modelo, o corpo de emagrecimento e morte ocorrer dentro de 4 semanas após a infecção. Dependente do Cepas de Salmonella usado para infecção, alguns ratos não podem sobreviver mais de 6 meses. <img src="/files/ftp_upload/1947/1947fig1.jpg" alt="Figura 1" /> Figura 1. Salmonella intestinal na espécie Salmonella aparecem malva (rosa ao roxo) na cor, devido às diferenças metabólicas na presença de cromógenos selecionado. Outras bactérias são inibido ou produzir colônias azul-esverdeada ou incolor. <img src="/files/ftp_upload/1947/1947fig2.jpg" alt="Figura 2" /> Figura 2. Proporções de sobrevivência de camundongos infectados com Salmonella cepa mutante PhoP c, c PhoP Avra, e PhoP AvrA-/AvrA c +, c PhoP Avra durante 4 semanas (28 dias). Tabela peso corporal 1. De camundongos infectados com 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> controle </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> * Comparado ao grupo controle p &lt;0,05 ** Quando comparado ao grupo controle p &lt;0,01 Tabela 2. Cepas de Salmonella utilizados neste estudo. <table border="1"><tbody><tr><td> Nome </td><td> Descrição </td><td> De referência ou fonte </td></tr><tr><td> Salmonella 14028s </td><td> Do tipo selvagem S. typhimurium </td><td> ATCC </td></tr><tr><td> PhoP c </td><td> Não-patogênicos complexo regulador mutante </td><td> Miller et al., 1990 </td></tr><tr><td> PhoP c Avra- </td><td> Avra mutação </td><td> Collier-Hyams et al., 2002 </td></tr><tr><td> PhoP c + AvrA-/AvrA </td><td> PhoP c Avra com Avra codificação complementada plasmídeo </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>

Não há conflitos de interesse declarados.

Para utilizar este sistema, é necessário que o pesquisador para aprender a gavagem os animais. Detalhamos uma metodologia para preparar a cultura bacteriana e gavagem a camundongos. Também mostramos como monitorar a persistência de Salmonella no trato gastrointestinal (TGI). Os passos críticos neste protocolo, incluindo: <ol><li> Estreptomicina pré-tratamento: Estreptomicina pré-tratamento, poderia se livrar de alguns comensais da flora intestinal e fazer os ratos suscetíveis à infecção por S...

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chronic salmonella infected mouse model

O modelo do rato infectado de bactérias é um sistema poderoso modelo para o estudo de áreas tais como inflamação, infecção, imunologia, transdução de sinal, e tumorigênese. Muitos pesquisadores têm se aproveitado da colite induzida por<em&gt; Salmonella</em&gt; Typhimurium para os estudos sobre a fase inicial de inflamação e infecção. No entanto, poucos relatos são a infecção crônica<em&gt; In vivo</em&gt;. Camundongos com<em&gt; Salmonella</em&gt; Existência persistente no trato gastrointestinal nos permitem explorar a host-bacteriana interação a longo prazo, transdução de sinal, e tumorigênese. Nós estabelecemos um modelo de camundongo bacteriana crônica infectados com<em&gt; Salmonella</em&gt; Typhimurium colonização no intestino do rato mais de 6 meses. Para utilizar este sistema, é necessário que o pesquisador para aprender a preparar a cultura bacteriana e sonda os animais. Detalhamos uma metodologia para cultura bacteriana preparar e ratos gavagem. Também mostramos como detectar a<em&gt; Salmonella</em&gt; Persistência no trato gastrointestinal. Globalmente, este protocolo vai ajudar os pesquisadores usando o modelo de camundongo infectado de bactérias para tratar fundamentalmente importantes questões biológicas e microbiológicas.

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

Chronic Salmonella Infected Mouse Model

Estabelecer um modelo de camundongo bacteriana crônica infectados com persistente<em&gt; Salmonella</em&gt; Typhimurium colonização no intestino durante 27 semanas.

Crônico Salmonella Modelo de camundongos infectados

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 visualizações.  University of Rochester. Estabelecer um modelo de camundongo bacteriana crônica infectados com persistente Salmonella Typhimurium colonização no intestino durante 27 semanas.

Vídeo: 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 rato ortotópico de câncer colorretal

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

Biologia

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 Ocular Modelo Hipertensão crônica em ratos 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.

Imuno-histoquímica em parafina da epiderme do rato utilizando anticorpos 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.

In vivo de imagens e tratamentos terapêuticos em um modelo de camundongo ortotópico de câncer de ovário

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.

Geração de camundongos derivadas de células-tronco pluripotentes induzidas

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.

Manter o Aedes aegypti , mosquitos infectados com 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.

O murino colina deficiente, Ethionine-completada (CDE) dieta modelo de lesão hepática crônica

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 pilha injeção maciça de células-tronco embrionárias leva a taxas mais elevadas 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;

Quasi-metagenomic análise de Salmonella de alimentos e amostras ambientais

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.