Name: visualization of il-22-expressing lymphocytes using reporter mice
Uploaded: 2017-01-25T12:30:00
Description: Watch this Scientific Journal Video about Visualization of IL-22-expressing Lymphocytes Using Reporter Mice at JoVE.com

We thank Kelli Czarra and Megan Karwan for animal technical assistance, Kathleen Noer and Roberta Matthai for flow cytometry assistance, and Donna Butcher andMiriam R. Anver for pathology analysis. This project was supported by a grant from the Ely and Edythe Broad Foundation (to Scott Durum) and has been funded in whole or in part with federal funds from the National Cancer Institute, National Institutes of Health, under Contract No. HHSN261200800001E (MRA).

Alle dieren kregen de juiste zorg in overeenstemming met de in de Guide 2011 voor de zorg en het gebruik van het Laboratorium Comité Dieren van Frederick National Laboratory for Cancer Research experimentele procedures. 1. Generatie van IL-22-tdTomato Reporter muizen door BAC recombineering OPMERKING: De muizen moeten onbewust en niet bewegen in reactie op een schadelijke prikkel. Steriliseer de chirurgische gebied met 70% ethanol en steriliseer alle chirurgische ...

<ol>
	<li>Awasthi, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cutting+edge:+IL-23+receptor+gfp+reporter+mice+reveal+distinct+populations+of+IL-17-producing+cells.">Cutting edge: IL-23 receptor gfp reporter mice reveal distinct populations of IL-17-producing cells.</a> J Immunol. 182, 5904-5908 (2009).</li><li>Price, A. E., Reinhardt, R. L., Liang, H. E., Locksley, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Marking+and+quantifying+IL-17A-producing+cells+in+vivo.">Marking and quantifying IL-17A-producing cells in vivo.</a> PLoS One. 7, e39750 (2012).</li><li>Kamanaka, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+of+interleukin-10+in+intestinal+lymphocytes+detected+by+an+interleukin-10+reporter+knockin+tiger+mouse.">Expression of interleukin-10 in intestinal lymphocytes detected by an interleukin-10 reporter knockin tiger mouse.</a> Immunity. 25, 941-952 (2006).</li><li>Sonnenberg, G. F., Fouser, L. A., Artis, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Border+patrol:+regulation+of+immunity,+inflammation+and+tissue+homeostasis+at+barrier+surfaces+by+IL-22.">Border patrol: regulation of immunity, inflammation and tissue homeostasis at barrier surfaces by IL-22.</a> Nat Immunol. 12, 383-390 (2011).</li><li>Dumoutier, L., Louahed, J., Renauld, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cloning+and+characterization+of+IL-10-related+T+cell-derived+inducible+factor+(IL-TIF),+a+novel+cytokine+structurally+related+to+IL-10+and+inducible+by+IL-9.">Cloning and characterization of IL-10-related T cell-derived inducible factor (IL-TIF), a novel cytokine structurally related to IL-10 and inducible by IL-9.</a> J Immunol. 164, 1814-1819 (2000).</li><li>Cella, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+human+natural+killer+cell+subset+provides+an+innate+source+of+IL-22+for+mucosal+immunity.">A human natural killer cell subset provides an innate source of IL-22 for mucosal immunity.</a> Nature. 457, 722-725 (2009).</li><li>Sanos, S. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RORgammat+and+commensal+microflora+are+required+for+the+differentiation+of+mucosal+interleukin+22-producing+NKp46++cells.">RORgammat and commensal microflora are required for the differentiation of mucosal interleukin 22-producing NKp46+ cells.</a> Nat Immunol. 10, 83-91 (2009).</li><li>Spits, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Innate+lymphoid+cells--a+proposal+for+uniform+nomenclature.">Innate lymphoid cells--a proposal for uniform nomenclature.</a> Nat Rev Immunol. 13, 145-149 (2013).</li><li>Mazzucchelli, R. I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualization+and+identification+of+IL-7+producing+cells+in+reporter+mice.">Visualization and identification of IL-7 producing cells in reporter mice.</a> PLoS One. 4, e7637 (2009).</li><li>Shen, W., Hixon, J. A., McLean, M. H., Li, W. Q., Durum, S. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=IL-22-Expressing+Murine+Lymphocytes+Display+Plasticity+and+Pathogenicity+in+Reporter+Mice.">IL-22-Expressing Murine Lymphocytes Display Plasticity and Pathogenicity in Reporter Mice.</a> Front Immunol. 6, 662 (2015).</li><li>Gong, S., Yang, X. W., Li, C., Heintz, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Highly+efficient+modification+of+bacterial+artificial+chromosomes+(BACs)+using+novel+shuttle+vectors+containing+the+R6Kgamma+origin+of+replication.">Highly efficient modification of bacterial artificial chromosomes (BACs) using novel shuttle vectors containing the R6Kgamma origin of replication.</a> Genome Res. 12, 1992-1998 (2002).</li><li>Shen, W., Huang, Y., Tang, Y., Liu, D. P., Liang, C. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+general+method+to+modify+BACs+to+generate+large+recombinant+DNA+fragments.">A general method to modify BACs to generate large recombinant DNA fragments.</a> Mol Biotechnol. 31, 181-186 (2005).</li><li>Cho, A., Haruyama, N., Kulkarni, A. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+transgenic+mice.">Generation of transgenic mice.</a> Curr Protoc Cell Biol. Chapter. Chapter 19, Unit 19 11 (2009).</li><li>Danneman, P. J., Stein, S., Walshaw, S. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Humane+and+practical+implications+of+using+carbon+dioxide+mixed+with+oxygen+for+anesthesia+or+euthanasia+of+rats.">Humane and practical implications of using carbon dioxide mixed with oxygen for anesthesia or euthanasia of rats.</a> Lab Anim Sci. 47, 376-385 (1997).</li><li>Omata, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+coccidian+enteroepithelial+stages+of+Toxoplasma+gondii+from+the+intestinal+mucosa+of+cats+by+Percoll+density-gradient+centrifugation.">Isolation of coccidian enteroepithelial stages of Toxoplasma gondii from the intestinal mucosa of cats by Percoll density-gradient centrifugation.</a> Parasitol Res. 83, 574-577 (1997).</li><li>Kumar, N., Borth, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flow-cytometry+and+cell+sorting:+an+efficient+approach+to+investigate+productivity+and+cell+physiology+in+mammalian+cell+factories.">Flow-cytometry and cell sorting: an efficient approach to investigate productivity and cell physiology in mammalian cell factories.</a> Methods. 56, 366-374 (2012).</li><li>Fischer, A. H., Jacobson, K. A., Rose, J., Zeller, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cryosectioning+tissues.">Cryosectioning tissues.</a> CSH Protoc. 2008, pdb.prot4991 (2008).</li><li>Hilbe, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+automated+cellular+imaging+system+and+manual+microscopy+for+immunohistochemically+stained+cryostat+sections+of+lung+cancer+specimens+applying+p53,+ki-67+and+p120.">Comparison of automated cellular imaging system and manual microscopy for immunohistochemically stained cryostat sections of lung cancer specimens applying p53, ki-67 and p120.</a> Oncol Rep. 10, 15-20 (2003).</li><li>Suzuki, Y., Furukawa, M., Abe, J., Kashiwagi, M., Hirose, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Localization+of+porcine+trappin-2+(SKALP/elafin)+in+trachea+and+large+intestine+by+in+situ+hybridization+and+immunohistochemistry.">Localization of porcine trappin-2 (SKALP/elafin) in trachea and large intestine by in situ hybridization and immunohistochemistry.</a> Histochem Cell Biol. 114, 15-20 (2000).</li><li>Wolk, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=IL-22+increases+the+innate+immunity+of+tissues.">IL-22 increases the innate immunity of tissues.</a> Immunity. 21, 241-254 (2004).</li><li>Brand, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=IL-22+is+increased+in+active+Crohn's+disease+and+promotes+proinflammatory+gene+expression+and+intestinal+epithelial+cell+migration.">IL-22 is increased in active Crohn's disease and promotes proinflammatory gene expression and intestinal epithelial cell migration.</a> Am J Physiol Gastrointest Liver Physiol. 290, G827-G838 (2006).</li><li>Nagalakshmi, M. L., Rascle, A., Zurawski, S., Menon, S., Waal Malefyt, d. e., R, <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interleukin-22+activates+STAT3+and+induces+IL-10+by+colon+epithelial+cells.">Interleukin-22 activates STAT3 and induces IL-10 by colon epithelial cells.</a> Int Immunopharmacol. 4, 679-691 (2004).</li><li>Maloy, K. J., Powrie, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intestinal+homeostasis+and+its+breakdown+in+inflammatory+bowel+disease.">Intestinal homeostasis and its breakdown in inflammatory bowel disease.</a> Nature. 474, 298-306 (2011).</li><li>Andoh, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interleukin-22,+a+member+of+the+IL-10+subfamily,+induces+inflammatory+responses+in+colonic+subepithelial+myofibroblasts.">Interleukin-22, a member of the IL-10 subfamily, induces inflammatory responses in colonic subepithelial myofibroblasts.</a> Gastroenterology. 129, 969-984 (2005).</li><li>Bhan, A. K., Mizoguchi, E., Smith, R. N., Mizoguchi, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Colitis+in+transgenic+and+knockout+animals+as+models+of+human+inflammatory+bowel+disease.">Colitis in transgenic and knockout animals as models of human inflammatory bowel disease.</a> Immunol Rev. 169, 195-207 (1999).</li><li>Mombaerts, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spontaneous+development+of+inflammatory+bowel+disease+in+T+cell+receptor+mutant+mice.">Spontaneous development of inflammatory bowel disease in T cell receptor mutant mice.</a> Cell. 75, 274-282 (1993).</li><li>Powrie, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immune+regulation+in+the+intestine:+a+balancing+act+between+effector+and+regulatory+T+cell+responses.">Immune regulation in the intestine: a balancing act between effector and regulatory T cell responses.</a> Ann N Y Acad Sci. 1029, 132-141 (2004).</li><li>Ahlfors, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=IL-22+fate+reporter+reveals+origin+and+control+of+IL-22+production+in+homeostasis+and+infection.">IL-22 fate reporter reveals origin and control of IL-22 production in homeostasis and infection.</a> J Immunol. 193, 4602-4613 (2014).</li></ol>

IL-22 speelt een essentiële rol in de aangeboren afweer en weefsel remodeling. IL-22-producerende cellen zijn geïdentificeerd ex vivo door intracellulaire kleuring. Echter, het blijft moeilijk om IL-22 expressie volgen in situ, in de normale toestand of ontstekingsaandoeningen. Dit protocol beschrijft een nieuwe werkwijze voor een IL-22 reporter muismodel, waardoor we de reporter tot expressie brengen in vivo te lokaliseren ontwikkelen. Het reportergen codeert TdTomato werd in de IL-22 locus...

Celtype-specifieke expressie van reportergenen nuttig om cellen actief expressie het doelwit weefsels onder homeostatische en verstoorde staten identificeren. Het staat ook voor de zuivering van deze cellen die levensvatbaar blijven hun andere eigenschappen te bestuderen. Reportermuizen zijn gebruikt in het ophelderen van het werkingsmechanisme van bepaalde cytokines, transcriptiefactoren en regulerende elementen. Previous strategieën 1, 2, 3 hebben grotendeels gebruikt kloppen de reporter in het doel-locus in de muis chromosoom, een tijdrovende en kostbare procedure. Dus een eenvoudiger werkwijze voor het genereren van reportermuizen gewenst. Cytokinen zijn een brede klasse van kleine, uitgescheiden eiwitten / peptiden die immuunreacties door middel van intercellulaire signalering reguleren. Interleukine 22 (IL-22) is een cytokine met vele gerapporteerde activiteiten, zoals barrière functies, weefselherstel, en ontsteking 4. Alhoewel IL-22 werd oorspronkelijk ontdekt als een T-celproduct 5, vervolgrapporten toonde de expressie ervan in natural killer (NK) cellen bij de mens en muizen 6 7 en in andere klassen van lymfocyten aangeboren 8. Ondanks uitgebreid observatie van IL-22-producerende cellen visualisatie van IL-22 voorheen ex vivo stimulatie en permeabilisatie vlekken met antilichamen. Daarom zouden nieuwe IL-22 reportermuizen bijzonder nuttig om de functie van IL-22 in homeostatische en pathogene processen onderzoeken. Hier hebben we een vereenvoudigde reporter transgene muismodel voor de IL-22-producerende cellen te observeren in vivo en in vitro. Met behulp van een BAC recombineering methode 9, we ingevoegd tdTomato de cDNA sequentie met poly A signaal fragmenten in de IL-22 locons en vervangen exon 1. De andere onvertaalde regio, exons en regulatoire elementen werden niet verstoord, want we willen de natuurlijke regulatie van IL-22 zo goed mogelijk na te bootsen. De plaats van insertie reporter verstoort de signaalsequentie, resulterend in de accumulatie van de reporter in de cellen, in tegenstelling tot IL-22 zelf, die snel wordt uitgescheiden. Deze nieuwe werkwijze kan ook worden toegepast voor het genereren van reportermuizen voor andere uitgescheiden eiwitten.

<table border="0" cellpadding="0" cellspacing="0" width="775">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:164px;">RP23-401E11 BAC</td>
			<td style="width:151px;">Thermo Fisher Scientific</td>
			<td style="width:116px;">RPCI23.C</td>
			<td style="width:345px;">Need gene ID: 50929</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:164px;">NucleoBond BAC 100</td>
			<td style="width:151px;">Takara Clontech</td>
			<td style="width:116px;">740579</td>
			<td style="width:345px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:164px;">PCR SuperMix High Fidelity</td>
			<td style="width:151px;">Thermo Fisher Scientific</td>
			<td style="width:116px;">10790020</td>
			<td style="width:345px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:164px;">PI-SceI</td>
			<td style="width:151px;">New England Biolabs</td>
			<td style="width:116px;">R0696S</td>
			<td style="width:345px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:164px;">SpeI</td>
			<td style="width:151px;">New England Biolabs</td>
			<td style="width:116px;">R0133S</td>
			<td style="width:345px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:164px;">LB Broth</td>
			<td>Thermo Fisher Scientific</td>
			<td style="width:116px;">10855-001</td>
			<td style="width:345px;">1L: 10 g SELECT Peptone 140, 5 g SELECT Yeast Extract, 5 g sodium chloride&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:164px;">Anti-mouse CD3</td>
			<td style="width:151px;">eBioscience</td>
			<td style="width:116px;">11-0031</td>
			<td style="width:345px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:164px;">Anti-mouse CD4</td>
			<td style="width:151px;">eBioscience</td>
			<td style="width:116px;">17-0041</td>
			<td style="width:345px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:164px;">Anti-mouse CD45</td>
			<td>Thermo Fisher Scientific</td>
			<td style="width:116px;">MCD4530</td>
			<td style="width:345px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:164px;">Anti-mouse CD45RB</td>
			<td style="width:151px;">eBioscience</td>
			<td style="width:116px;">11-0455</td>
			<td style="width:345px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:164px;">Anti-mouse RFP</td>
			<td style="width:151px;">Abcam</td>
			<td style="width:116px;">Ab62341</td>
			<td style="width:345px;"></td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;width:164px;">HBSS, no calcium, no magnesium, no phenol red</td>
			<td style="width:151px;">Thermo Fisher Scientific</td>
			<td style="width:116px;">14175145</td>
			<td style="width:345px;">KCl, KH2PO4, Na2HPO4, NaHCO3, NaCl, D-Glucose</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:164px;">Dnase I</td>
			<td style="width:151px;">Roche</td>
			<td style="width:116px;">10104159001</td>
			<td style="width:345px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:164px;">ACK lysing buffer</td>
			<td style="width:151px;">Thermo Fisher Scientific</td>
			<td style="width:116px;">A1049201</td>
			<td style="width:345px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:164px;">Percoll</td>
			<td style="width:151px;">GE healthcare life sciences</td>
			<td style="width:116px;">17-0891-01</td>
			<td style="width:345px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:164px;">Collagenase D</td>
			<td style="width:151px;">Roche</td>
			<td style="width:116px;">11088858001</td>
			<td style="width:345px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:164px;">Dispase II (neutral protease, grade II)</td>
			<td style="width:151px;">Roche</td>
			<td style="width:116px;">4942078001</td>
			<td style="width:345px;"></td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;width:164px;">IX70 inverted fluorescence microscope</td>
			<td style="width:151px;">Olympus</td>
			<td style="width:116px;"></td>
			<td style="width:345px;">Ask for quote</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:164px;">Nikon Eclipse 80i microscope</td>
			<td style="width:151px;">Nikon</td>
			<td style="width:116px;"></td>
			<td style="width:345px;">Ask for quote</td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;width:164px;">Dynal shaker</td>
			<td style="width:151px;">Electron Microscopy Science</td>
			<td style="width:116px;">61050-10</td>
			<td style="width:345px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:164px;">FACSAria</td>
			<td style="width:151px;">BD Bioscience</td>
			<td style="width:116px;"></td>
			<td style="width:345px;">Ask for quote</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:164px;">LSRII SORP/flow cytometry</td>
			<td style="width:151px;">Becton, Dickinson and Company&#160;</td>
			<td style="width:116px;"></td>
			<td style="width:345px;">Ask for quote</td>
		</tr>
	</tbody>
</table>

visualization of il-22-expressing lymphocytes using reporter mice

Reportermuizen zijn op grote schaal gebruikt voor de lokalisatie van de expressie van gerichte genen observeren. Dit protocol richt zich op een strategie om een ​​nieuwe transgene reporter muismodel vast te stellen. We kozen te visualiseren interleukine (IL) 22 genexpressie omdat cytokine heeft belangrijke activiteiten in de darm, waar het bijdraagt ​​aan weefsel beschadigd door ontsteking herstellen. Reportersystemen bieden grote voordelen boven andere werkwijzen voor het identificeren producten in vivo. In het geval van IL-22, had andere studies eerste geïsoleerde cellen uit weefsels en vervolgens opnieuw gestimuleerde cellen in vitro. IL-22, die normaal wordt uitgescheiden, werd opgesloten in cellen met een geneesmiddel, en intracellulaire kleuring werd gebruikt voor het visualiseren. Deze werkwijze identificeert cellen die in staat is IL-22, maar niet bepalen of ze dat deden in vivo. De reporter ontwerp omvat het inbrengen van een gen voor een fluorescerend eiwit (tdTomato) in de IL-22-gen in dergelijke way dat het fluorescerende eiwit kan worden uitgescheiden en derhalve blijft opgesloten in de cellen in vivo. Fluorescerende producenten kunnen vervolgens worden gevisualiseerd in weefselcoupes of door ex vivo analyse door middel van stromingscytometrie. De eigenlijke bouwproces voor de reporter opgenomen recombineering een bacterieel kunstmatig chromosoom dat de IL-22-gen bevatte. Deze gemanipuleerde chromosoom werd vervolgens in het muis genoom. Homeostatische IL-22 reporter expressie werd waargenomen in verschillende muizenweefsels, waaronder de milt, thymus, lymfeklieren, Peyer&#39;s patch en darmen, met stroomcytometrie analyse. Colitis werd geïnduceerd door T-cellen (CD4 + CD45RBhigh) overdracht en reporter expressie werd gevisualiseerd. Positieve T cellen werden eerst in de mesenterische lymfeklieren en vervolgens geaccumuleerd zij in de lamina propria van de distale dunne darm en colon weefsel. De strategie van het gebruik van BAC&#39;s gaf goede-fidelity reporter uitdrukking in vergelijking met IL-22 expressie en het is eenvoudiger dan knock-in procedures.

Een muis-IL-22 reporter transgen is gemaakt met recombineering een bacterieel kunstmatig chromosoom die het IL-22 locus wijzigen. Figuur 1 een schema van pBACe3.6 vector die het gen sacBII een positieve selectiemerker en chlooramfenicol antibioticumresistentiegen 11. Na de introductie tdTomato in exon 1, werd de signaalpeptidesequentie verstoord, zie figuur 2. Aldus werd de tdTomato reporter opgesloten in de IL-22 tot exp...

Watch this Scientific Journal Video about Visualization of IL-22-expressing Lymphocytes Using Reporter Mice at JoVE.com

Visualization of IL-22-expressing Lymphocytes Using Reporter Mice

We describe here a transgenic reporter mouse model to visualize the IL-22-producing cells inside different mouse tissues. This method can be used to track the location of other cytokines or secretary proteins in the mouse.

Visualisatie van IL-22-uiting van lymfocyten Met behulp van Reporter Muizen

Reporter mice have been widely used to observe the localization of expression of targeted genes. This protocol focuses on a strategy to establish a new ...

Research

JoVE Journal

Immunology and Infection

Visualization of Bacterial Toxin Induced Responses Using Live Cell Fluorescence Microscopy

Visualisatie van bacteriële toxine geïnduceerde responsen met Live Cell Fluorescentie Microscopie

Bacterial toxins bind to cholesterol in membranes, forming pores that allow for leakage of cellular contents and influx of materials from the external ...

Isolation of Lymphocytes from Mouse Genital Tract Mucosa

Isolatie van Lymfocyten van Mouse geslachtsorganen Mucosa

Mucosal surfaces, including in the gastrointestinal, urogenital, and respiratory tracts, provide portals of entry for pathogens, such as viruses and ...

Isolation and Th17 Differentiation of Na&iuml;ve CD4 T Lymphocytes

Isolation and Th17 Differentiation of NaÃƒÆ’Ã‚Â¯ve CD4 T Lymphocytes

Isolatie en Th17 Differentiatie van naïeve CD4 T-lymfocyten

Th17 cells are a distinct subset of T cells that have been found to produce interleukin 17 (IL-17), and differ in function from the other T cell subsets ...

Long Term Intravital Multiphoton Microscopy Imaging of Immune Cells in Healthy and Diseased Liver Using CXCR6.Gfp Reporter Mice

Long Term intravitale Multiphoton Microscopie Beeldvorming van immuuncellen in gezonde en zieke lever behulp CXCR6.Gfp Reporter Muizen

Liver inflammation as a response to injury is a highly dynamic process involving the infiltration of distinct subtypes of leukocytes including monocytes, ...

Intravital Imaging of Neutrophil Priming Using IL-1&#946; Promoter-driven DsRed Reporter Mice

Intravital Imaging of Neutrophil Priming Using IL-1ÃƒÅ½Ã‚Â² Promoter-driven DsRed Reporter Mice

Intravitale Imaging van neutrofielen Priming gebruik van IL-1β-Promoter gedreven DsRed Reporter Muizen

Neutrophils are the most abundant leukocytes in human blood circulation and are quickly recruited to inflammatory sites. Priming is a critical event that ...

Isolation and Activation of Murine Lymphocytes

Isolatie en Activering van Muizen lymfocyten

B and T cells, with their extremely diverse antigen-receptor repertoires, have the ability to mount specific immune responses against almost any invading ...

Visualization of Biofilm Formation in Candida albicans Using an Automated Microfluidic Device

Visualization of Biofilm Formation in Candida albicans Using an Automated Microfluidic Device

Visualisatie van Biofilm vorming in Candida albicans met behulp van een geautomatiseerd Microfluidic-apparaat

Candida albicans is the most common fungal pathogen of humans, causing about 15% of hospital-acquired sepsis cases. A major virulence attribute of C. ...

Imaging Mycobacterium tuberculosis in Mice with Reporter Enzyme Fluorescence

Imaging Mycobacterium tuberculosis in Mice with Reporter Enzyme Fluorescence

Mycobacterium tuberculosis in muizen met verslaggever enzym fluorescentie Imaging

Reporter enzyme fluorescence (REF) utilizes substrates that are specific for enzymes present in target organisms of interest for imaging or detection by ...

Intravital Imaging of Intraepithelial Lymphocytes in Murine Small Intestine

Intravital beeldvorming van epitheliale lymfocyten in muizen dunne darm

Intraepithelial lymphocytes expressing &#947;&#948; T cell receptor (&#947;&#948; IEL) play a key role in immune surveillance of the intestinal ...

Stem Cell-Derived Viral Ag-Specific T Lymphocytes Suppress HBV Replication in Mice

Stamcel afkomstige virale AG-specifieke T-lymfocyten onderdrukken HBV-replicatie bij muizen

Hepatitis B virus (HBV) infection is a global health issue. With over 350 million people affected worldwide, HBV infection remains the leading cause of ...