Name: cell aggregation assays to evaluate the binding of the drosophila notch with trans-ligands and its inhibition by cis-ligands
Uploaded: 2018-01-02T14:00:00
Description: Watch this Scientific Journal Video about Cell Aggregation Assays to Evaluate the Binding of the Drosophila Notch with Trans-Ligands and its Inhibition by Cis-Ligands at JoVE.com

Forfatterne anerkender støtte fra NIH/NIGMS (R01GM084135 til HJN) og Mizutani grundlag for Glycoscience (grant #110071 til HJN), og er taknemmelig for, at Tom V. Lee for diskussioner og forslag på assays, og Spyros Artavanis-Tsakonas, Hugo Bell, Robert Fleming, Ken Irvine og Drosophila genomforskning ressource Center (DGRC) for plasmider og cellelinjer.

1. forberedelse af dobbelt strandede RNA (dsRNA) for Shams Knockdown
<ol>
	<li>PCR-amplifikation af produkter
	<ol>
		<li>Bruge wild-type gul hvid (y w) genomisk DNA og pAc5.1-EGFP som skabelon, og de følgende primer par til at forstærke DNA fragmenter bruges i dsRNA syntese. Brug følgende PCR termisk profil: denaturering (95 ° C, 30 s), udglødning (58 ° C, 30 s) og filtypenavn (72 ° C, 1 min). 
		Forbedret grøn fluorescerende proteiner (EGFP<...

<ol>
	<li>de Celis, J. F., Bray, S., Garcia-Bellido, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Notch+signalling+regulates+veinlet+expression+and+establishes+boundaries+between+veins+and+interveins+in+the+Drosophila+wing.">Notch signalling regulates veinlet expression and establishes boundaries between veins and interveins in the Drosophila wing.</a> Development. 124 (10), 1919-1928 (1997).</li><li>Fortini, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Notch+signaling:+the+core+pathway+and+its+posttranslational+regulation.">Notch signaling: the core pathway and its posttranslational regulation.</a> Dev Cell. 16 (5), 633-647 (2009).</li><li>del Alamo, D., Rouault, H., Schweisguth, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanism+and+significance+of+cis-inhibition+in+Notch+signalling.">Mechanism and significance of cis-inhibition in Notch signalling.</a> Curr Biol. 21 (1), R40-R47 (2011).</li><li>Sprinzak, D., 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=Cis-interactions+between+Notch+and+Delta+generate+mutually+exclusive+signalling+states.">Cis-interactions between Notch and Delta generate mutually exclusive signalling states.</a> Nature. 465 (7294), 86-90 (2010).</li><li>Kopan, R., Ilagan, M. X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+canonical+Notch+signaling+pathway:+unfolding+the+activation+mechanism.">The canonical Notch signaling pathway: unfolding the activation mechanism.</a> Cell. 137 (2), 216-233 (2009).</li><li>Huppert, S. S., Jacobsen, T. L., Muskavitch, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Feedback+regulation+is+central+to+Delta-Notch+signalling+required+for+Drosophila+wing+vein+morphogenesis.">Feedback regulation is central to Delta-Notch signalling required for Drosophila wing vein morphogenesis.</a> Development. 124 (17), 3283-3291 (1997).</li><li>Lee, T. V., 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=Negative+regulation+of+notch+signaling+by+xylose.">Negative regulation of notch signaling by xylose.</a> PLoS Genet. 9 (6), e1003547 (2013).</li><li>Lee, T. V., Pandey, A., Jafar-Nejad, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Xylosylation+of+the+Notch+receptor+preserves+the+balance+between+its+activation+by+trans-Delta+and+inhibition+by+cis-ligands+in+Drosophila.">Xylosylation of the Notch receptor preserves the balance between its activation by trans-Delta and inhibition by cis-ligands in Drosophila.</a> PLoS Genet. 13 (4), e1006723 (2017).</li><li>Jacobsen, T. L., Brennan, K., Arias, A. M., Muskavitch, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cis-interactions+between+Delta+and+Notch+modulate+neurogenic+signalling+in+Drosophila.">Cis-interactions between Delta and Notch modulate neurogenic signalling in Drosophila.</a> Development. 125 (22), 4531-4540 (1998).</li><li>Schneider, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+lines+derived+from+late+embryonic+stages+of+Drosophila+melanogaster.">Cell lines derived from late embryonic stages of Drosophila melanogaster.</a> J Embryol Exp Morphol. 27 (2), 353-365 (1972).</li><li>Fehon, R. G., 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=Molecular+interactions+between+the+protein+products+of+the+neurogenic+loci+Notch+and+Delta,+two+EGF-homologous+genes+in+Drosophila.">Molecular interactions between the protein products of the neurogenic loci Notch and Delta, two EGF-homologous genes in Drosophila.</a> Cell. 61 (3), 523-534 (1990).</li><li>Fleming, R. J., 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=An+extracellular+region+of+Serrate+is+essential+for+ligand-induced+cis-inhibition+of+Notch+signaling.">An extracellular region of Serrate is essential for ligand-induced cis-inhibition of Notch signaling.</a> Development. 140 (9), 2039-2049 (2013).</li><li>Saj, 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=A+combined+ex+vivo+and+in+vivo+RNAi+screen+for+notch+regulators+in+Drosophila+reveals+an+extensive+notch+interaction+network.">A combined ex vivo and in vivo RNAi screen for notch regulators in Drosophila reveals an extensive notch interaction network.</a> Dev Cell. 18 (5), 862-876 (2010).</li><li>Fiuza, U. M., Klein, T., Martinez Arias, A., Hayward, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+of+ligand-mediated+inhibition+in+Notch+signaling+activity+in+Drosophila.">Mechanisms of ligand-mediated inhibition in Notch signaling activity in Drosophila.</a> Dev Dyn. 239 (3), 798-805 (2010).</li><li>Ahimou, F., Mok, L. P., Bardot, B., Wesley, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+adhesion+force+of+Notch+with+Delta+and+the+rate+of+Notch+signaling.">The adhesion force of Notch with Delta and the rate of Notch signaling.</a> J Cell Biol. 167 (6), 1217-1229 (2004).</li><li>Livak, K. J., Schmittgen, T. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+relative+gene+expression+data+using+real-time+quantitative+PCR+and+the+2(-Delta+Delta+C(T))+Method.">Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method.</a> Methods. 25 (4), 402-408 (2001).</li><li>Okajima, T., Xu, A., Irvine, K. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modulation+of+notch-ligand+binding+by+protein+O-fucosyltransferase+1+and+fringe.">Modulation of notch-ligand binding by protein O-fucosyltransferase 1 and fringe.</a> J Biol Chem. 278 (43), 42340-42345 (2003).</li><li>Acar, 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=Rumi+is+a+CAP10+domain+glycosyltransferase+that+modifies+Notch+and+is+required+for+Notch+signaling.">Rumi is a CAP10 domain glycosyltransferase that modifies Notch and is required for Notch signaling.</a> Cell. 132 (2), 247-258 (2008).</li><li>Bruckner, K., Perez, L., Clausen, H., Cohen, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glycosyltransferase+activity+of+Fringe+modulates+Notch-Delta+interactions.">Glycosyltransferase activity of Fringe modulates Notch-Delta interactions.</a> Nature. 406 (6794), 411-415 (2000).</li><li>Yamamoto, 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=A+mutation+in+EGF+repeat-8+of+Notch+discriminates+between+Serrate/Jagged+and+Delta+family+ligands.">A mutation in EGF repeat-8 of Notch discriminates between Serrate/Jagged and Delta family ligands.</a> Science. 338 (6111), 1229-1232 (2012).</li></ol>

Canonical Notch signalering afhænger af samspillet mellem Notch receptor og dens ligander5. Selv om de fleste undersøgelser på Notch pathway primært betragter binding af Notch og ligander i tilstødende celler (trans), hak og samme cellerække ligander interagerer, og disse såkaldte cis-interaktioner kan spille en hæmmende rolle i hak signalering3,4. Derfor for at dechifrere de underliggende virkningerne af en modif...

Canonical Notch signalering er en kortrækkende celle til celle kommunikationsmekanisme, der kræver fysisk kontakt mellem tilstødende celler for at lette samspillet mellem Notch receptorer og deres ligander1. Samspillet mellem Notch receptor (til stede på overfladen af signal-modtagende celler) og ligander (til stede på overfladen af signal at sende celler) indleder Notch signalering og er kendt som trans-aktivering2. På den anden side interaktion mellem Notch og dens ligander i den samme celle fører til hæmning af Notch pathway og er kendt som cis-hæmning3. Balance mellem trans- og cis-interaktioner er nødvendige for at sikre optimal ligand-afhængige Notch signalering4. Drosophila har en hak receptor og to ligander (Delta og Serrate) i modsætning til pattedyr, der har fire Notch receptorer og fem ligander [jagged 1 (JAG1), JAG2, delta-lignende 1 (DLL1), DLL3 og DLL4]5. At have denne enkelhed, tilbyder Drosophila model lethed til at dissekere/undersøgelse pathway modifikatorer virkninger på Notch-ligand interaktioner og efterfølgende på Notch signalering. I visse sammenhænge under dyrenes udvikling (herunder wing udvikling i Drosophila), både cis- og trans-interaktioner er involveret til at opnå ordentlig hak signalering og celle skæbne1,6 . Det er vigtigt at skelne virkninger af Notch pathway modifikatorer i disse sammenhænge på cis- og trans-interaktioner i hak med sin ligander.
Vores gruppe tidligere rapporteret at tilføjelsen af en kulhydrat rest kaldet xylose til Drosophila Notch negativt regulerer Notch signalering i visse sammenhænge, herunder wing udvikling7. Tab af shams (enzymet der xylosylates Notch) fører til en "tab af wing vene" fænotype7. Mere nylig, gen dosering eksperimenter og klonede analyse blev brugt til at vise, at tab af shams øger Delta-medieret Notch fokuseren. At skelne mellem om den forbedrede Notch signalering i shams mutanter er et resultat af nedsat cis-hæmning eller øget trans-aktivering, ektopiske overekspression undersøgelser af Notch ligander i larve wing fantasiens diske du bruger driveren, dpp-GAL4 blev udført. Disse eksperimenter leveres beviser tyder på, at Shams regulerer trans-aktivering af hak af Delta uden at påvirke Notch SNG-hæmning af ligander8. Dog feed-back bestemmelser og virkninger af endogene ligander kan komplicere fortolkningen af ektopiske overekspression undersøgelser1,6,9.
Du kan løse problemet, Drosophila S2 celler10 blev brugt, som giver en enkel in vitro- system for Notch-ligand interaktion undersøgelser11,12. S2 celler ikke udtrykke endogene Notch receptor og Delta ligand11 og udtrykke et lavt niveau af takkede13, som ikke påvirker Notch-ligand sammenlægning eksperimenter8. Derfor, S2 celler kan være stabilt eller forbigående transfekteret af hak og/eller individuelle ligander (Delta eller Serrate) til at generere celler, der udelukkende express Notch receptor eller en af dens ligander eller en kombination af disse. Blanding af hak-udtrykker S2 celler med ligand-udtrykker S2 celler resulterer i dannelsen af Heterotypiske aggregater medieret af receptor-ligand bindende11,12,14. Kvantitativ bestemmelse af den samlede dannelse giver et mål for trans-bindende mellem Notch og dens ligander15 (figur 1). Ligeledes S2 celler kan være co transfected med hak og Delta eller Serrate ligander (dvs. cis-ligander). CIS-ligander i cellernes Notch-udtrykker S2 ophæve sammenkædning af Notch med trans-ligander og resultere i nedsat samlede dannelse8,12,14. Den relative nedgang i samlede dannelse forårsaget af cis-ligander giver et mål for den hæmmende effekt af cis-ligander bindingen mellem Notch og trans-ligander (figur 2). I overensstemmelse hermed, celle sammenlægning assays blev udnyttet til at undersøge effekten af tab af xylosylation på trans- og cis-interaktioner mellem Notch og dens ligander.
Vi præsenterer her, en detaljeret protokol for celle sammenlægning assays til formål at evaluere binding af Notch med trans-ligander og dens hæmning af cis-ligander bruger Drosophila S2 celler. Som et eksempel, vi leverer de data, der tillod os at bestemme virkningen af Notch xylosylation bindingen mellem Notch og trans-Delta8. Disse enkle assays give en semi-kvantitative vurdering af hak-ligand interaktioner i vitro og hjælpe med at bestemme de molekylære mekanismer bag effekterne i vivo af Notch pathway modifikatorer.

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			<td height="22" style="height:23px;width:415px;">BioWhittaker Schneider&#8217;s Drosophila medium, Modified</td>
			<td style="width:187px;">Lonza</td>
			<td style="width:131px;">04-351Q</td>
			<td style="width:396px;"></td>
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		<tr height="19">
			<td height="19" style="height:19px;width:415px;">HyClone Penicillin-Streptomycin 100X solution</td>
			<td style="width:187px;">GE Healthcare lifescience&#160;</td>
			<td style="width:131px;">SV30010</td>
			<td style="width:396px;"></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:415px;">CELLSTAR 6 well plate</td>
			<td style="width:187px;">Greiner Bio-One</td>
			<td style="width:131px;">657 160</td>
			<td style="width:396px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:415px;">CELLSTAR 24 well plate</td>
			<td style="width:187px;">Greiner Bio-One</td>
			<td style="width:131px;">662160</td>
			<td style="width:396px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:415px;">VWR mini shaker</td>
			<td style="width:187px;">Marshell Sceintific</td>
			<td>12520-956</td>
			<td style="width:396px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:415px;">Hemocytometer</td>
			<td style="width:187px;">Fisher Sceintific&#160;</td>
			<td>267110</td>
			<td style="width:396px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:415px;">FuGENE HD Transfection Reagent</td>
			<td style="width:187px;">Promega</td>
			<td style="width:131px;">E2311</td>
			<td style="width:396px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:415px;">MEGAscrip T7 Transcription Kit</td>
			<td style="width:187px;">Ambion</td>
			<td style="width:131px;">AM1334</td>
			<td style="width:396px;"></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:415px;">Quick-RNA MiniPrep (RNA purification Kit)</td>
			<td style="width:187px;">Zymo Research</td>
			<td style="width:131px;">R1054</td>
			<td style="width:396px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:415px;">VistaVision Inverted microscope</td>
			<td>VWR</td>
			<td style="width:131px;"></td>
			<td style="width:396px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;width:415px;">9MP USB2.0 Microscope Digital Camera + Advanced Software</td>
			<td style="width:187px;">AmScope</td>
			<td style="width:131px;">MU-900</td>
			<td style="width:396px;">Image acquisition using ToupView software</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;width:415px;">PureLink Quick Gel Extraction Kit</td>
			<td style="width:187px;">Invitrogen</td>
			<td style="width:131px;">K210012</td>
			<td style="width:396px;"></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:415px;">Fetal Bovine Serum</td>
			<td style="width:187px;">GenDepot</td>
			<td style="width:131px;">F0600-050</td>
			<td style="width:396px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:415px;">Methotrexate</td>
			<td style="width:187px;">Sigma-Aldrich</td>
			<td style="width:131px;">A6770-10</td>
			<td style="width:396px;"></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:415px;">Hygromycin B</td>
			<td style="width:187px;">Invitrogen</td>
			<td style="width:131px;">HY068-L6</td>
			<td style="width:396px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:22px;width:415px;">Copper sulphate</td>
			<td style="width:187px;">Macron Fine Chemicals</td>
			<td style="width:131px;">4448-02</td>
			<td style="width:396px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:415px;">S2 cells</td>
			<td style="width:187px;">Invitrogen</td>
			<td style="width:131px;">R69007</td>
			<td style="width:396px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;width:415px;">S2-SerrateTom cells</td>
			<td style="width:187px;"></td>
			<td style="width:131px;"></td>
			<td style="width:396px;">Gift from R. Fleming (Fleming et al, Development, 2013)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:415px;">S2-Delta cells</td>
			<td style="width:187px;">DGRC</td>
			<td style="width:131px;">152</td>
			<td style="width:396px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:415px;">S2-Notch cells</td>
			<td style="width:187px;">DGRC</td>
			<td style="width:131px;">154</td>
			<td style="width:396px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:415px;">pMT-Delta vector</td>
			<td style="width:187px;">DGRC</td>
			<td style="width:131px;">1021</td>
			<td style="width:396px;">Gift from S. Artavanis-Tsakonas</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;width:415px;">pMT-Serrate vector</td>
			<td style="width:187px;"></td>
			<td style="width:131px;"></td>
			<td style="width:396px;">Gift from Ken Irvine (Okajima et al, JBC, 2003)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:415px;">pMT-Notch vector</td>
			<td style="width:187px;">DGRC</td>
			<td style="width:131px;">1022</td>
			<td style="width:396px;">Gift from S. Artavanis-Tsakonas</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:415px;">pAc5.1-EGFP</td>
			<td style="width:187px;"></td>
			<td style="width:131px;"></td>
			<td style="width:396px;">Gift from Hugo Bellen</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:415px;">TaqMan RNA-to-Ct 1-Step Kit</td>
			<td style="width:187px;">Applied Biosystem</td>
			<td style="width:131px;">1611091</td>
			<td style="width:396px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:415px;">TaqMan Gene Expression Assay for CG9996 (Shams)</td>
			<td style="width:187px;">Applied Biosystem</td>
			<td>Dm02144576_g1&#160;</td>
			<td style="width:396px;">with FAM-MGB dye</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:415px;">TaqMan Gene Expression Assay for CG7939 (RpL32)</td>
			<td style="width:187px;">Applied Biosystem</td>
			<td>Dm02151827_g1</td>
			<td style="width:396px;">with FAM-MGB dye</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:415px;">Applied Biosystems 7900HT Fast Real-Time PCR system</td>
			<td style="width:187px;">Applied Biosystem</td>
			<td style="width:131px;">4351405</td>
			<td style="width:396px;">96-well Block module</td>
		</tr>
	</tbody>
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cell aggregation assays to evaluate the binding of the drosophila notch with trans-ligands and its inhibition by cis-ligands

Notch signalering er en evolutionært bevarede celle-celle kommunikationssystem anvendes bredt i dyrenes udvikling og voksen vedligeholdelse. Samspillet mellem Notch receptor og ligander fra tilstødende celler inducerer aktivering af den signalvejen (trans-aktivering), mens interaktion med ligander fra den samme celle hæmmer signalering (cis-hæmning). Passende balance mellem trans-aktivering og cis-hæmning hjælper med at etablere optimale niveauer af Notch signalering i nogle sammenhænge under dyrenes udvikling. På grund af de overlappende udtryk domæner Notch og dens ligander i mange celletyper og eksistensen af feedback-mekanismer, at studere virkningerne af en given posttranslationel modifikation på trans- versus cis-vekselvirkninger mellem Notch og dens ligander i vivo er vanskeligt. Her, beskriver vi en protokol for Drosophila S2 celler i celle-sammenlægning assays til at vurdere virkningerne af bankede ned en hak pathway modifier for bindingen af hak til hver ligand i trans og i SNG-landene. S2 celler stabilt eller forbigående transfekteret med en kærv-udtrykker vektor er blandet med celler, der udtrykker hver Notch ligand (S2-Delta eller S2-savtakkede). Trans-bindingen mellem receptor og ligander resulterer i dannelsen af Heterotypiske celle aggregater og måles i forhold til antallet af aggregater pr. mL sammensat af &#62; 6 celler. At undersøge den hæmmende effekt af cis-ligander, S2 celler Co udtrykker Notch og hver ligand er blandet med S2-Delta eller S2-savtakkede celler og antallet af aggregater er kvantificeret som beskrevet ovenfor. Den relative nedgang i antallet af aggregater på grund af tilstedeværelsen af cis-ligander giver et mål for SNG-ligand-medieret hæmning af trans-bindende. Disse ligetil assays kan levere semi-kvantitative data om effekten af genetiske eller farmakologiske manipulationer på bindingen af hak til sit ligander, og kan hjælpe med at decifrere de molekylære mekanismer i vivo -effekten af sådanne manipulationer på Notch signalering.

Vores i vivo bemærkninger foreslog, at tab af xylosyltransferase gen shams resultater i gevinst på Notch signalering skyldes øget Delta-medieret trans-aktivering af Notch uden at påvirke cis-hæmning af Hak af ligander8. For at teste dette begreb, blev i vitro celle sammenlægning assays udført. Først, shams udtryk i S2 celler blev slået ved hjælp af shams dsRNA. EGFP dsRNA blev brugt s...

Watch this Scientific Journal Video about Cell Aggregation Assays to Evaluate the Binding of the Drosophila Notch with Trans-Ligands and its Inhibition by Cis-Ligands at JoVE.com

Cell Aggregation Assays to Evaluate the Binding of the Drosophila Notch with Trans-Ligands and its Inhibition by Cis-Ligands

I vivo systemers kompleksitet gør det vanskeligt at skelne mellem aktivering og hæmning af Notch receptor af trans- og cis-ligander, henholdsvis. Vi præsenterer her, en protokol baseret på in vitro- celle-sammenlægning assays til kvalitative og semi-kvantitative evaluering af bindingen af Drosophila hak til trans-ligander vs cis-ligander.

Celle sammenlægning Assays til at evaluere bindingen af Drosophila Notch med Trans-ligander og dens hæmning af Cis-ligander

Notch signaling is an evolutionarily conserved cell-cell communication system used broadly in animal development and adult maintenance. Interaction of the ...

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Developmental Biology

Cell Aggregation Assays to Evaluate the Binding of the Drosophila Notch with Trans-Ligands and its Inhibition by Cis-Ligands

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Many parallels exist between the Drosophila and mammalian hematopoietic systems, even though Drosophila lack the lymphoid lineage that characterize ...

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Notch signaling is a key component of multiple physiological and pathological processes. The nature of Notch signaling, however, makes in vitro ...

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We have developed a cell-based assay using Drosophila cells that recapitulates apical constriction initiated by folded gastrulation (Fog), a secreted ...