Name: intraductal injection of lps as a mouse model of mastitis: signaling visualized via an nf-&kappa;b reporter transgenic
Uploaded: 2012-09-04T12:00:00
Description: Watch this Scientific Journal Video about Intraductal Injection of LPS as a Mouse Model of Mastitis: Signaling Visualized via an NF-ÃŽÂºB Reporter Transgenic at JoVE.com

Dette arbejde blev finansieret af NIH tilskud CA113734 tildelt F Yull og Department of Veterans Affairs (TS Blackwell).

Alle dyreforsøg blev godkendt af Vanderbilt University Institutional Animal Care og Use Udvalg. 1. Fremstilling af transgene mus <ol><li> Hunmus, der var positive for NGL (N F-KB-G FP-L uciferase) reporter-transgen (beskrevet tidligere 1) anvendes til denne procedure. Når kvinder er mindst 6 uger gamle, parre dem med en FVB vildtype han. Harem avl kan anvendes (op til 4 hunner i et bur med en mandlig). (Bemærk: FVB-stamme mus blev anvendt til den foreliggende...

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	<li>Everhart, M. B., Han, W., Sherrill, T. P., Arutiunov, M., Polosukhin, V. V., Burke, J. R., Sadikot, R. T., Christman, J. W., Yull, F. E., Blackwell, T. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Duration+and+intensity+of+NF-&#954;B+activity+determine+the+severity+of+endotoxin-induced+acute+lung+injury.">Duration and intensity of NF-&#954;B activity determine the severity of endotoxin-induced acute lung injury.</a> The Journal of Immunology. 176, 4995-5005 (2006).</li><li>Notebaert, S., Carlsen, H., Janssen, D., Vandenabeele, P., Blomhoff, R., Meyer, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+imaging+of+NF-&#206;&#186;B+activity+during+Escherichia+coli-induced+mammary+gland+infection.">In vivo imaging of NF-&#206;&#186;B activity during Escherichia coli-induced mammary gland infection.</a> Cellular Microbiology. 10, 1249-1258 (2008).</li><li>Elazar, S., Gonen, E., Livneh-Kol, A., Rosenshine, I., Shpigel, N. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neutrophil+recruitment+in+endotoxin-induced+murine+mastitis+is+strictly+dependent+on+mammary+alveolar+macrophages.">Neutrophil recruitment in endotoxin-induced murine mastitis is strictly dependent on mammary alveolar macrophages.</a> Veterinary Research. 41, 10 (2010).</li><li>Connelly, L., Barham, W., Pigg, R., Sait-Jean, L., Sherrill, T., Cheng, D. S., Chodosh, L. A., Blackwell, T. S., Yull, F. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activation+of+nuclear+factor+kappaB+in+mammary+epithelium+promotes+milk+loss+during+mammary+development+and+infection.">Activation of nuclear factor kappaB in mammary epithelium promotes milk loss during mammary development and infection.</a> J. Cell. Physiol. 222, 73-81 (2010).</li><li>Yardeni, T., Eckhaus, M., Morris, H. D., Huizing, M., Hoogstraten-Miller, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retro-orbital+injections+in+mice.">Retro-orbital injections in mice.</a> Lab Animal. 40, 155-160 (2011).</li><li>Plante, I., Stewart, M. K., Laird, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+mammary+gland+development+and+function+in+mouse+models.">Evaluation of mammary gland development and function in mouse models.</a> J. Vis. Exp. (21), e2828 (2011).</li><li>Behbod, F., Kittrell, F. S., LaMarca, H., Edwards, D., Kerbawy, S., Heestand, J. C., Young, E., Mukhopadhyay, P., Yeh, H., Allred, D. C., Hu, M., Polyak, K., Rosen, J. M., Medina, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+intraductal+human-in-mouse+transplantation+model+mimics+the+subtypes+of+ductal+carcinoma+in+situ.">An intraductal human-in-mouse transplantation model mimics the subtypes of ductal carcinoma in situ.</a> Breast Cancer Research. 11, R66 (2009).</li><li>Zheng, J., Watson, A. D., Kerr, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome-wide+expression+analysis+of+lipopolysaccharide-induced+mastitis+in+a+mouse+model.">Genome-wide expression analysis of lipopolysaccharide-induced mastitis in a mouse model.</a> Infection and Immunity. 74, 1907-1915 (2006).</li><li>Nguyen, D., Beeman, N., Lewis, M., Schaack, J., Neville, M. C., Ip, M. M., Asch, 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=.">.</a> Intraductal Injection into the Mouse Mammary Gland. Methods in Mammary Gland Biology and Breast Cancer Research. , 259-270 (2000).</li><li>Lee, J. W., Paape, M. J., Zhao, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recombinant+bovine+soluble+CD14+reduces+severity+of+experimental+Escherichia+coli+mastitis+in+mice.">Recombinant bovine soluble CD14 reduces severity of experimental Escherichia coli mastitis in mice.</a> Veterinary Research. 34, 307-316 (2003).</li><li>Elazar, S., Gonen, E., Livneh-Kol, A., Rosenshine, I., Shpigel, N. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Essential+role+of+neutrophils+but+not+mammary+alveolar+macrophages+in+a+murine+model+of+acute+Escherichia+coli+mastitis.">Essential role of neutrophils but not mammary alveolar macrophages in a murine model of acute Escherichia coli mastitis.</a> Veterinary Research. 41, 53 (2010).</li><li>Bannerman, D. D., Paape, M. J., Hare, W. R., Sohn, E. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Increased+levels+of+LPS-binding+protein+in+bovine+blood+and+milk+following+bacterial+lipopolysaccharide+challenge.">Increased levels of LPS-binding protein in bovine blood and milk following bacterial lipopolysaccharide challenge.</a> Journal of Dairy Science. 86, 3128-3137 (2003).</li><li>Gonen, E., Vallon-Eberhard, A., Elazar, S., Harmelin, A., Brenner, O., Rosenshine, I., Jung, S., Shpigel, N. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Toll-like+receptor+4+is+needed+to+restrict+the+invasion+of+Escherichia+coli+P4+into+mammary+gland+epithelial+cells+in+a+murine+model+of+acute+mastitis.">Toll-like receptor 4 is needed to restrict the invasion of Escherichia coli P4 into mammary gland epithelial cells in a murine model of acute mastitis.</a> Cellular Microbiology. 9, 2826-2838 (2007).</li><li>Notebaert, S., Demon, D., Vanden Berghe, ., Vandenabeele, T., P, ., Meyer, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inflammatory+mediators+in+Escherichia+coli-induced+mastitis+in+mice.">Inflammatory mediators in Escherichia coli-induced mastitis in mice.</a> Comparative Immunology, Microbiology, and Infectious Diseases. 31, 551-565 (2008).</li><li>Osterman, K. L., Rahm, V. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lactation+mastitis:+bacterial+cultivation+of+breast+milk+symptoms,+treatment,+and+outcome.">Lactation mastitis: bacterial cultivation of breast milk symptoms, treatment, and outcome.</a> Journal of Human Lactation. 16, 297-302 (2000).</li><li>Sordillo, L. M., Streicher, K. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mammary+Gland+Immunity+and+Mastitis+Susceptibility.">Mammary Gland Immunity and Mastitis Susceptibility.</a> Journal of Mammary Gland Biology and Neoplasia. 7, 135-146 (2002).</li><li>Blackwell, T. S., Yull, F. E., Chen, C. L., Venkatakrishnan, A., Blackwell, T. R., Hicks, D. J., Lancaster, L. H., Christman, J. W., Kerr, L. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+genetically+altered+mice+to+investigate+the+role+of+nuclear+factor-kappa+B+activation+and+cytokine+gene+expression+in+sepsis-induced+ARDS.">Use of genetically altered mice to investigate the role of nuclear factor-kappa B activation and cytokine gene expression in sepsis-induced ARDS.</a> Chest. 116, 73S-74S (1999).</li><li>Stathopoulos, G. T., Sherrill, T. P., Cheng, D. S., Scoggins, R. M., Han, W., Polosukhin, V. V., Connelly, L., Yull, F. E., Fingleton, B., Blackwell, T. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epithelial+NF-kappaB+activation+promotes+urethane-induced+lung+carcinogenesis.">Epithelial NF-kappaB activation promotes urethane-induced lung carcinogenesis.</a> Proceedings of the National Academy of Sciences. 104, 18514-18519 (2007).</li><li>Blackwell, T. S., Hipps, A. N., Yamamoto, Y., Han, W., Barham, W. J., Ostrowski, M. C., Yull, F. E., Prince, L. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NF-&#954;B+signaling+in+fetal+lung+macrophages+disrupts+airway+morphogenesis.">NF-&#954;B signaling in fetal lung macrophages disrupts airway morphogenesis.</a> The Journal of Immunology. 187, 2740-2747 (2011).</li><li>Chiang, S. H., Bazuine, M., Lumeng, C. N., Geletka, L. M., Mowers, J., White, N. M., Ma, J. T., Zhou, J., Qi, N., Westscott, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+protein+kinase+IKKepsilon+regulates+energy+balance+in+obese+mice.">The protein kinase IKKepsilon regulates energy balance in obese mice.</a> Cell. 138, 961-975 (2009).</li><li>Singh, M. V., Kapoun, A., Higgins, L., Kutschke, W., Thurman, J. M., Zhang, R., Singh, M., Yang, J., Guan, X., Lowe, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ca2+/calmodulin-dependent+kinase+II+triggers+cell+membrane+injury+by+inducing+complement+factor+B+gene+expression+in+the+mouse+heart.">Ca2+/calmodulin-dependent kinase II triggers cell membrane injury by inducing complement factor B gene expression in the mouse heart.</a> The Journal of Clinical Investigation. 119, 986-996 (2009).</li></ol>

Her udnytter vi en transgen NF-KB reporter mus og en optimeret injektionsteknik til model mastitis, betændelse i brystvæv oftest forårsaget af bakteriel infektion. Det mest kritiske skridt i denne procedure er den intraductal injektion selv. I praksis er det normalt klart, om intraductal injektion har været vellykket. Injektionen af ​​midlerne (LPS eller PBS) skal føles fluid og der bør være lidt eller ingen modstand som sprøjten tømmes. Den mest almindelige faldgrube er beskadi...

<table border="1"><tbody><tr><td> Navn på reagenset </td><td> Firma </td><td> Katalognummer </td><td> Kommentarer </td></tr><tr><td> Lipopolysaccharid fra Escherichia coli (LPS) </td><td> Sigma </td><td> L 2880 </td><td> serotype 055: B5 </td></tr><tr><td> PBS </td><td> Mediatech, Inc. </td><td> 46 til 013-CM </td><td></td></tr><tr><td> 31 Gauge insulinsprøjte </td><td> Becton Dickinson </td><td> 328.438 </td><td> Kort nål </td></tr><tr><td> Tang </td><td> Verden Precision Instruments </td><td> 15.908 </td><td></td></tr><tr><td> Dissektionsmikroskop </td><td> Leica </td><td> M3Z </td><td></td></tr><tr><td> Terrell Isofluran, USP </td><td> MINRAD INC. for Rx Elite </td><td> NDC 66794-011-25 </td><td></td></tr><tr><td> IVIS 200 billeddannelsessystem &lt;/ Td&gt; <td> Caliper (tidligere Xenogen) </td><td> N / A </td><td></td></tr><tr><td> Sprøjte til luciferin injektion </td><td> Becton Dickinson </td><td> 309.597 </td><td> 1cc, 26G5 / 8 </td></tr><tr><td> D-luciferin Firefly-natriumsalt-monohydrat (Luciferin substrat) </td><td> Biosynth kemi og biologi </td><td> L-8240 </td><td> Fortyndes til 10 mg / ml </td></tr></tbody></table>

intraductal injection of lps as a mouse model of mastitis: signaling visualized via an nf-&kappa;b reporter transgenic

Dyremodeller for human sygdom er nødvendige for at strengt studere stadier af sygdommen og dermed forbundne mekanismer, og i sidste ende, som prækliniske modeller til at teste indgreb. Ved disse fremgangsmåder beskriver vi en teknik, hvor lipopolysaccharid (LPS) injiceres i lakterende muse brystkirtel via brystvorten, effektiv modellering mastitis eller inflammation af kirtlen. Denne simulerede infektion resulterer i forøget nuclear factor kappa B (NF-KB) signalering, som visualiseres ved bioluminescerende billeddannelse af en NF-KB luciferase reporter mus 1. Vores ultimative mål med at udvikle disse metoder var at undersøge inflammation associeret med mastitis i lakterende kirtel, der ofte inkluderer rødme, hævelse, og immun celleinfiltration 2,3. Derfor var vi meget opmærksomme at snit eller nogen form for sårdannelse på huden, brystvorten eller kirtlen for at indføre LPS kunneikke anvendes i vore metoder, da den fremgangsmåde sandsynligvis ville forvirre den read-out af inflammation. Vi har også ønsket en straight-forward metode, der ikke kræver specielt fremstillet håndtegnede pipetter eller anvendelse af mikromanipulatorer at holde disse specialiserede værktøjer på plads. Derfor besluttede vi at bruge et kommercielt tilgængeligt insulin sprøjte og at injicere midlet i den mammae kanal af et intakt nippel. Denne metode har været vellykket og tilladt os at studere betændelse i forbindelse med LPS-injektion uden yderligere virkninger overlejret med fremgangsmåden ifølge injektion. Desuden denne fremgangsmåde anvendes et NF-KB luciferase reporter transgen mus og bioluminescerende imaging-teknologi til visuelt og kvantitativt viser øget NF-KB signalering inden for LPS-injicerede kirtel 4. Disse metoder er af interesse for forskere af mange discipliner, der ønsker at model sygdom i diegivende brystkirtel, som i sidste ende, at teknikkenbeskrevet her kan anvendes til injektion af en række stoffer, og er ikke begrænset til kun LPS.

Watch this Scientific Journal Video about Intraductal Injection of LPS as a Mouse Model of Mastitis: Signaling Visualized via an NF-ÃŽÂºB Reporter Transgenic at JoVE.com

Intraductal Injection of LPS as a Mouse Model of Mastitis: Signaling Visualized via an NF-ÃƒÅ½Ã‚ÂºB Reporter Transgenic

Beskrevet her er en teknik, hvor lipopolysaccharid injiceres i lakterende muse brystkirtel via brystvorten at simulere mastitis, en betingelse, der almindeligvis forårsaget af bakteriel infektion. Lipopolysaccharid injektion resulterer i øget nuclear factor kappa B (NF-KB) signalering, visualiseret ved bioluminescerende billeddannelse af en NF-KB luciferase reporter mus.

Intraductal Injektion af LPS som en musemodel af Mastitis: Signaling visualiseres via en NF-KB Reporter Transgenic

Animal models of human disease are necessary in order to rigorously study stages of disease progression and associated mechanisms, and ultimately, as ...

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Intraductal Injection of LPS as a Mouse Model of Mastitis: Signaling Visualized via an NF-&kappa;B Reporter Transgenic

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