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 protokoller ble gjennomgått og godkjent av Institutional Animal Care og bruk Committee (IACUC) ved Texas A &amp; M University College of Dentistry. 1. husdyravl <ol><li> Bruk tre dyremodeller i denne studien. For å undersøke skjebnen til embryonale chondrocytes i knokkelen formasjon, første gangs bruk Col10a1-Cre 15 mus og krysse dem med Rosa26 tdTomato (B6, 129S6- Gt (ROSA) 26Sor TM9 (CAG-tdTomato) Hze / J) mus for å f?...

<ol>
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På grunn av teknologiske begrensninger, er det alltid vanskelig å undersøke oppførselen til celler in vivo. Imidlertid blir celle avstamning tracing teknikk vist seg å være et kraftig verktøy for å studere cellebiologi 7-9. I denne studien har vi ytterligere forbedre denne protokollen ved å kombinere den med immunfluorescens. På denne måte kan celle skjebne defineres av flere beslektede markører, som utvider anvendelsen av avstamning sporing. Videre viser denne samlokalisering av immunflu...

Under utviklingen utgjør endochondral bendannelse for over 80% av skjelettvolum. Det er en utbredt oppfatning at det begynner med apoptose av hypertrofiske chondrocytes. Deretter ble cellene fra den underliggende benmarg invadere og sette i gang angiogenese, etterfulgt av nytt ben avsetning av ben marrow- og periosteum-avledede celler 1,2. Cellen skjebne hypertrofiske chondrocytes (HC&#39;er), derimot, har vært et problem for debatt i flere tiår 3. Til å begynne med ble HC&#39;er ansett for å være den ende av kondrocytt differensiering vei, og apoptose ble generelt antatt å være den endelige skjebnen til HC&#39;er. Nå, noen forskere antyder at i det minste noen HC&#39;er kunne overleve og bidra til endochondral beindannelse. Selv om de foreslås at veksten plate chondrocytter hadde evnen til å transdifferentiate inn i osteoblaster basert på ultrastruktur, immunhistokjemisk farging, og in vitro studier 46 Ingen av disse metodene were definitive demonstrere chondrocyttransplantasjon bidrag til osteoblast avstamning. Cellen avstamning tracing teknikk gir en mer grundig måte å studere celle skjebne. I korthet sett, en rekombinase enzym, som bare uttrykkes i et bestemt type celler, stimulerer ekspresjonen av reportergenet. På denne måten blir denne type celle og deres etterkommere permanent merket 7. Den Cre-loxP system er vanligvis brukes i avstamning sporing. Cre (den recombinase enzym) vil eksisere STOP sekvens mellom de to loxP-seter og aktivere reporter i en bestemt cellelinje (figur 1A). I noen tilfeller kan undersøkeren velge et gunstig tidspunkt for å aktivere Cre ved hjelp av et stoff, slik som tamoxifen, forårsaker Cre til å smelte til en modifisert form av østrogenreseptoren (Cre ERT2) 8. Fluorescent journalister har blitt standard i avstamning tracing eksperimenter fordi de dramatisk redusere kompleksitetenog forbedre nøyaktigheten og effektiviteten av celle skjebne sporing 8,9. tdTomato blir det beste valget blant fluorescerende reportere siden det har den lyseste fluorescerende protein og den sterkeste epifluorescence, noe som gjør det enkelt visualisert 7 (figur 1A). Ved hjelp av Rosa26 tdTomato avstamning sporing system, har vår gruppe og andre forskere vist at HC&#39;er kan endre sin fenotype til beinceller under utvikling 10-14. For å oppnå dette, har vi utviklet to sett med tracing kombinasjoner med Rosa26 tdtomato (allestedsnærværende ekspresjon i alle celler) / Cre (spesifikk til kondrocytter) mus: 2.3Col1a1-GFP (spesifikk til osteoblaster) og immunfluorescens (spesifikk til benceller). Dataene viser at begge metoder er levedyktige måter å studere celle skjebne 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

Cellen avstamning tracing system har vært brukt hovedsakelig i utviklingsbiologi studier. Bruken av Cre rekombinase gjør det mulig for aktivering av reporter i en spesifikk cellelinje og alt avkom. Her brukte vi cellen avstamning tracing teknikk for å vise at chondrocytes direkte forvandle seg til osteoblaster og osteocytter under lange bein og kjeve knokkelen utvikling ved hjelp av to typer Cre, Col10a1-Cre og aggrecan-Cre ERT2 (Agg-Cre ERT2), krysset med Rosa26 tdTomato. Både Col10 og aggrecan er godt anerkjent markører for chondrocytes. På bakgrunn av dette, har vi utviklet en ny metode-celle avstamning sporing i forbindelse med fluorescerende immunhistokjemi å definere celle skjebne ved å analysere uttrykket av spesifikke cellemarkører. Runx2 (en markør for tidlig stadium osteogene celler) og Dentin matrise protein1 (DMP1, en markør for sent stadium osteogene celler) varanvendes for å identifisere kondrocytt-avledede beinceller og deres differensiering status. Denne kombinasjonen er ikke bare utvider anvendelsen av cellen avstamning tracing, men forenkler også generering av sammensatte mus. Enda viktigere er de nummer, beliggenhet, og differensierings statusene foreldrecellen avkom vises samtidig, noe som gir mer informasjon enn celle avstamning sporing alene. I konklusjonen, co-anvendelse av cellen avstamning sporing teknikker og immunfluorescens er et kraftig verktøy for å undersøke cellebiologi in vivo.

Chondrocytes Direkte Transform til beinceller (osteoblaster og osteocytter) i Mandibular knokkelen og lange ben. Aggrecan, en kritisk genet for chondrogenesis, er hovedsakelig uttrykt i tidlige og modne chondrocytes 18. Som et resultat av injeksjon av tamoxifen på 2 uker gamle i Agg-Cre ERT2; Rosa26 tdTomato mus aktivert røde tomat reporter i alle c...

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

Vi utviklet to sett med sporing kombinasjoner i Rosa26 tdtomato (overalt uttrykt i alle celler) / Cre (spesielt uttrykt i chondrocytes) mus: en med 2.3Col1a1-GFP (spesifikke for osteoblaster) og ett med immunfluorescens (spesifikke for beinceller). Dataene viser den direkte transformasjon av kondrocytter inn i beinceller.

Samlokalisering av Cell Lineage Markører og tomat 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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Cell Lineage Analyses and Gene Function Studies Using Twin-spot MARCM

Cell Lineage Analyser og gen-funksjon studier med Twin-spot MARCM

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 av protein-protein-interaksjoner og samlokalisering mellom komponenter av gap-, tett- og adherens-junks i murine mammarykjertler

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 av humaniserte benmarg mikromiljøer i mus og deres visualisering av levende bildebehandling

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

Metoder for Imaging intracellulær pH i hårsekken stamcelleforskningen slektslinje i Live Drosophila Ovarian vev

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

Direkte avstamning omprogrammering av voksen mus Fibroblast å Erythroid Progenitors

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

Isotropic lys-ark mikroskopi og automatisert Cell Lineage analyser til katalog Caenorhabditis elegans embryogenesis med Subcellulære oppløsning

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