Name: co-localization of cell lineage markers and the tomato signal
Uploaded: 2016-12-28T14:00:00
Description: Watch this Scientific Journal Video about Co-localization of Cell Lineage Markers and the Tomato Signal at JoVE.com

This study was supported by NIH grant DE025014 to JQF.

Alle protocollen zijn beoordeeld en goedgekeurd door de Institutional Animal Care en gebruik Comite (IACUC) aan de Texas A &amp; M University College of Dentistry goedgekeurd. 1. Animal Breeding <ol><li> Met drie diermodellen in deze studie. Om het lot van de embryonale chondrocyten in condyl formatie te onderzoeken, het eerste gebruik Col10a1-Cre 15 muizen en kruis ze met Rosa26 tdTomato (B6; 129S6- Gt (ROSA) 26Sor tm9 (CAG-tdTomato) Hze</s...

<ol>
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Door de technologische beperkingen, is het altijd moeilijk om het gedrag van cellen in vivo te onderzoeken. Echter, de cellijn tracing techniek blijkt een krachtig hulpmiddel voor het bestuderen van celbiologie 7-9 zijn. In deze studie hebben we verder verbeteren dit protocol door het te combineren met immunofluorescentie. Zo kunnen lot van de cel worden gedefinieerd door meerdere gerelateerde markers, die de toepassing van lineage tracing verbreedt. Bovendien is deze co-lokalisatie van immunofluores...

Tijdens de ontwikkeling endochondrale botvorming goed voor meer dan 80% van het skelet volume. Het wordt algemeen aangenomen dat het begint met de apoptose van hypertrofische chondrocyten. Vervolgens worden de cellen van het onderliggende beenmerg invasie en angiogenese initiëren, gevolgd door afzetting van nieuw bot beenmerg en periosteum-afgeleide cellen 1,2. Het lot van de cel van hypertrofische chondrocyten (HC), echter, is een kwestie van debat voor decennia 3. Aanvankelijk werden HC&#39;s als het einde van de chondrocyte differentiatieomzettingsweg zijn en apoptose werd algemeen aangenomen dat het uiteindelijke lot van HC zijn. Nu sommige onderzoekers suggereren dat ten minste enkele HCs kunnen overleven en bijdragen aan botvorming. Hoewel ze de groei voorgestelde plaat chondrocyten had de mogelijkheid om transdifferentiëren in osteoblasten basis van ultrastructuur, immunohistochemische kleuring en in vitro studies 46, geen van deze werkwijzen were definitief bij het aantonen van chondrocyte bijdrage aan de osteoblast lineage. De cel lineage tracing techniek zorgt voor een meer rigoureuze manier om lot van de cel te bestuderen. Kort gesproken, een recombinase enzym dat alleen in een bepaald type cel tot expressie wordt gebracht, stimuleert de expressie van het reportergen. Zo worden dergelijke cellen en hun nakomelingen permanente etikettering 7. De Cre-loxP systeem wordt vaak gebruikt in lineage tracing. Cre (de recombinase enzym) zal de STOP sequentie tussen de twee loxP plaatsen accijnzen en activeer de verslaggever in een bepaalde cellijn (Figuur 1A). In sommige gevallen kan de onderzoeker kiezen een gunstig tijdstip te Cre activeren met een geneesmiddel, zoals tamoxifen, waardoor Cre te fuseren met een gewijzigde vorm van de oestrogeenreceptor (Cre ERT2) 8. TL-reporters hebben de standaard in lineage tracing experimenten omdat ze drastisch de complexiteit te verminderen gewordenen het verbeteren van de nauwkeurigheid en efficiëntie van de cel lot traceren 8,9. tdTomato is steeds de beste keuze onder de tl-reporters omdat het de helderste fluorescerend eiwit en de sterkste epifluorescentie, waardoor het gemakkelijk gevisualiseerd 7 (Figuur 1A). Via de Rosa26 tdTomato lineage tracing, onze fractie en andere onderzoekers hebben aangetoond dat HC fenotype in botcellen tijdens de ontwikkeling 10-14 kunnen veranderen. Om dit te bereiken, hebben wij twee traceren combinaties met Rosa26 tdtomato (alomtegenwoordige expressie in alle cellen) / Cre (specifiek voor chondrocyten) muizen: 2.3Col1a1-GFP (specifiek voor osteoblasten) en immunofluorescentie (specifiek voor botcellen). De gegevens tonen dat beide methoden levensvatbaar manieren lot van de cel te bestuderen in vivo.

<table border="0" cellpadding="0" cellspacing="0" width="1099">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Tamoxifen&#160;</td>
			<td style="width:220px;">Sigma</td>
			<td style="width:136px;">T5648</td>
			<td style="width:496px;">activate the Cre event&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Paraformaldehyde</td>
			<td>Sigma&#160;</td>
			<td>P6148</td>
			<td>fix the sample</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ethylenediaminetetraacetic acid</td>
			<td>Alfa Aesar</td>
			<td>A10713</td>
			<td>decalcify the hard tissue</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sucrose&#160;</td>
			<td>Sigma</td>
			<td>S0389</td>
			<td>dehydrate the tissue</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hyaluronidase from bovine testes&#160;</td>
			<td>Sigma</td>
			<td>H4272</td>
			<td>retrieve antigen for immunochemical staining</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Bovine serum albumin</td>
			<td>Sigma&#160;</td>
			<td>A3059</td>
			<td>blocking solution&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">primary antibody for Runx2&#160;</td>
			<td>Cell Signal</td>
			<td>D1L7F</td>
			<td>primary antibody for immunochemical staining</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">primary antibody for DMP1</td>
			<td style="width:220px;">provided by Dr. Chunlin Qin</td>
			<td style="width:136px;"></td>
			<td>primary antibody for immunochemical staining</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">anti-rabbit IgG</td>
			<td style="width:220px;">Sigma</td>
			<td style="width:136px;">18140</td>
			<td>control for immunochemical staining</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">secondary antibody&#160;</td>
			<td>Invitrogen</td>
			<td>A11008</td>
			<td>second antibody for immunochemical staining</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">OCT</td>
			<td>Tissue-Tek</td>
			<td>4583</td>
			<td>embed the sample for frozen section</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tween 20</td>
			<td>Fisher Scientific</td>
			<td>BP337</td>
			<td>PBST</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">non-fluorescing antifade mountant</td>
			<td>Life technologies</td>
			<td>P36934</td>
			<td>mounting slides</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DAPI</td>
			<td>Life technologies</td>
			<td>P36931</td>
			<td>nuclear staining</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hydrophobic Barrier Pen</td>
			<td>Vector Laboratories</td>
			<td></td>
			<td>circle the section on the slide for for immunochemical staining&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Xylazine</td>
			<td style="width:220px;">AnaSed</td>
			<td></td>
			<td>anesthetization&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ketaset&#160;</td>
			<td>Zoetis</td>
			<td></td>
			<td>anesthetization&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">cryosection machine</td>
			<td style="width:220px;">Leica</td>
			<td style="width:136px;">CM1860 UV</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">confocal microscope</td>
			<td style="width:220px;">Leica</td>
			<td>DM6000 CFS&#160;</td>
			<td></td>
		</tr>
	</tbody>
</table>

co-localization of cell lineage markers and the tomato signal

De cel lineage tracing systeem is voornamelijk gebruikt in ontwikkelingsbiologie studies. Het gebruik van Cre recombinase maakt de activering van de reporter in een specifieke cellijn en al het nageslacht. Hier hebben we gebruik gemaakt van de cel lineage tracing techniek aan te tonen dat chondrocyten direct in osteoblasten en osteocyten transformeren tijdens lange botten en kaakkopje ontwikkeling met behulp van twee soorten Cre, Col10a1-Cre en aggrecan-Cre ERT2 (Agg-Cre ERT2), gekruist met Rosa26 tdTomato. Zowel Col10 en aggrecan zijn goed herkend markers voor chondrocyten. Op basis hiervan hebben we een nieuwe methode-cellijn tracing in combinatie met fluorescerende immunohistochemie te definiëren lot van de cel door het analyseren van de expressie van specifieke celmarkers. Runx2 (een marker voor een vroeg stadium osteogene cellen) en Dentin matrix protein1 (DMP1, een marker voor een laat stadium osteogene cellen) warengebruikt om chondrocyte afgeleide botcellen en hun differentiatie status identificeren. Deze combinatie verbreedt niet alleen de toepassing van cellijn tracing, maar vereenvoudigt ook de vorming van verbinding muizen. Belangrijker is het aantal, de locatie en differentiatie status van oudercel nageslacht gelijktijdig worden weergegeven, die meer informatie dan alleen cellijn tracing. Kortom, de co-toepassing van cellijn tracing technieken en immunofluorescentie is een krachtig hulpmiddel voor het onderzoeken van celbiologie in vivo.

Chondrocyten Direct Transform in botcellen (Osteoblasten en Osteocyten) in de kaakkop en Long Bone. Aggrecan, een kritische gen voor chondrogenese, voornamelijk in de vroege en volwassen chondrocyten 18. Dientengevolge, de injectie van tamoxifen op 2 weken in Agg-Cre ERT2; Rosa26 tdTomato muizen geactiveerd de rode tomaten reporter in alle chondrocyte...

Watch this Scientific Journal Video about Co-localization of Cell Lineage Markers and the Tomato Signal at JoVE.com

Co-localization of Cell Lineage Markers and the Tomato Signal

Wij ontwikkelden twee traceren combinaties Rosa26 tdtomato (alomtegenwoordig tot expressie in alle cellen) / Cre (specifiek tot expressie in chondrocyten) muizen: één met 2.3Col1a1-GFP (specifiek voor osteoblasten) en een met immunofluorescentie (specifiek voor botcellen). De gegevens tonen de directe omzetting van chondrocyten in botcellen.

Co-localisatie van Cell Lineage Markers en de tomaat Signal

The cell lineage tracing system has been used predominantly in developmental biology studies. The use of Cre recombinase allows for the activation of the ...

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Mosaic analysis with a repressible cell marker (MARCM) is a positive mosaic labeling system that has been widely applied in Drosophila neurobiological ...

Analysis of Protein-protein Interactions and Co-localization Between Components of Gap, Tight, and Adherens Junctions in Murine Mammary Glands

Analyse van eiwit-eiwitinteracties en co-lokalisatie tussen componenten van kloof-, strakke- en adherentie-knooppunten in muizenmammarysklieren

Cell-cell interactions play a pivotal role in preserving tissue integrity and the barrier between the different compartments of the mammary gland. These ...

Bioengineering of Humanized Bone Marrow Microenvironments in Mouse and Their Visualization by Live Imaging

Bioengineering van Humanized Botmarg Microenvironments in Mouse en hun visualisatie door Live Imaging

Human hematopoietic stem cells (HSCs) reside in the bone marrow (BM) niche, an intricate, multifactorial network of components producing cytokines, growth ...

Methods for Imaging Intracellular pH of the Follicle Stem Cell Lineage in Live Drosophila Ovarian Tissue

Methods for Imaging Intracellular pH of the Follicle Stem Cell Lineage in Live Drosophila Ovarian Tissue

Methoden voor Imaging intracellulaire pH van de follikel stamcel overdrachtslijn in Live Drosophila eierstokweefsel

Changes in intracellular pH (pHi) play important roles in the regulation of many cellular functions, including metabolism, proliferation, and ...

Direct Lineage Reprogramming of Adult Mouse Fibroblast to Erythroid Progenitors

Directe bloedlijn herprogrammering van volwassen muis Fibroblast te Erythroid voorlopercellen

Erythroid cell commitment and differentiation proceed through activation of a lineage-restricted transcriptional network orchestrated by a group of cell ...

Isotropic Light-Sheet Microscopy and Automated Cell Lineage Analyses to Catalogue Caenorhabditis elegans Embryogenesis with Subcellular Resolution

Isotrope licht-vel microscopie en geautomatiseerde Cell Lineage analyses catalogus Caenorhabditis embryogenese met subcellulaire resolutie

Caenorhabditis elegans (C. elegans) stands out as the only organism in which the challenge of understanding the cellular origins of an entire nervous ...