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 blev revideret og godkendt af Institutional Animal Care og brug Udvalg (IACUC) ved Texas A &amp; M University College of Dentistry. 1. Husdyravl <ol><li> Bruge tre dyremodeller i denne undersøgelse. For at undersøge, hvilken skæbne de embryonale chondrocytter i condylus dannelse, første brug Col10a1-Cre 15 mus og krydse dem med Rosa26 tdTomato (B6; 129S6- Gt (ROSA) 26Sor TM9 (CAG-tdTomato) Hze / J) mus for at ...

<ol>
	<li>Gibson, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Active+role+of+chondrocyte+apoptosis+in+endochondral+ossification.">Active role of chondrocyte apoptosis in endochondral ossification.</a> Microsc Res Tech. 43 (2), 191-204 (1998).</li><li>Kronenberg, H. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Developmental+regulation+of+the+growth+plate.">Developmental regulation of the growth plate.</a> Nature. 423 (6937), 332-336 (2003).</li><li>Shapiro, I. M., Adams, C. S., Freeman, T., Srinivas, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fate+of+the+hypertrophic+chondrocyte:+microenvironmental+perspectives+on+apoptosis+and+survival+in+the+epiphyseal+growth+plate.">Fate of the hypertrophic chondrocyte: microenvironmental perspectives on apoptosis and survival in the epiphyseal growth plate.</a> Birth Defects Res C Embryo Today. 75 (4), 330-339 (2005).</li><li>Kahn, A. J., Simmons, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chondrocyte-to-osteocyte+transformation+in+grafts+of+perichondrium-free+epiphyseal+cartilage.">Chondrocyte-to-osteocyte transformation in grafts of perichondrium-free epiphyseal cartilage.</a> Clin Orthop Relat Res. (129), 299-304 (1977).</li><li>Roach, H. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Trans-differentiation+of+hypertrophic+chondrocytes+into+cells+capable+of+producing+a+mineralized+bone+matrix.">Trans-differentiation of hypertrophic chondrocytes into cells capable of producing a mineralized bone matrix.</a> Bone Miner. 19 (1), 1-20 (1992).</li><li>Park, 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=Dual+pathways+to+endochondral+osteoblasts:+a+novel+chondrocyte-derived+osteoprogenitor+cell+identified+in+hypertrophic+cartilage.">Dual pathways to endochondral osteoblasts: a novel chondrocyte-derived osteoprogenitor cell identified in hypertrophic cartilage.</a> Biol Open. 4 (5), 608-621 (2015).</li><li>Kretzschmar, K., Watt, F. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lineage+tracing.">Lineage tracing.</a> Cell. 148 (1-2), 33-35 (2012).</li><li>Humphreys, B. D., DiRocco, D. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lineage-tracing+methods+and+the+kidney.">Lineage-tracing methods and the kidney.</a> Kidney Int. 86 (3), 481-488 (2014).</li><li>Romagnani, P., Rinkevich, Y., Dekel, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+use+of+lineage+tracing+to+study+kidney+injury+and+regeneration.">The use of lineage tracing to study kidney injury and regeneration.</a> Nat Rev Nephrol. 11 (7), 420-431 (2015).</li><li>Yang, 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=Osteogenic+fate+of+hypertrophic+chondrocytes.">Osteogenic fate of hypertrophic chondrocytes.</a> Cell Res. 24 (10), 1266-1269 (2014).</li><li>Yang, L., Tsang, K. Y., Tang, H. C., Chan, D., Cheah, K. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hypertrophic+chondrocytes+can+become+osteoblasts+and+osteocytes+in+endochondral+bone+formation.">Hypertrophic chondrocytes can become osteoblasts and osteocytes in endochondral bone formation.</a> Proc Natl Acad Sci U S A. 111 (33), 12097-12102 (2014).</li><li>Jing, 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=Chondrocytes+Directly+Transform+into+Bone+Cells+in+Mandibular+Condyle+Growth.">Chondrocytes Directly Transform into Bone Cells in Mandibular Condyle Growth.</a> J Dent Res. 94 (12), 1668-1675 (2015).</li><li>Zhou, X., von der Mark, K., Henry, S., Norton, W., Adams, H., de Crombrugghe, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chondrocytes+transdifferentiate+into+osteoblasts+in+endochondral+bone+during+development,+postnatal+growth+and+fracture+healing+in+mice.">Chondrocytes transdifferentiate into osteoblasts in endochondral bone during development, postnatal growth and fracture healing in mice.</a> PLoS Genet. 10 (12), (2014).</li><li>Purcell, P., Trainor, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Mighty+Chondrocyte:+No+Bones+about+It.">The Mighty Chondrocyte: No Bones about It.</a> J Dent Res. 94 (12), 1625-1627 (2015).</li><li>Gebhard, 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=Specific+expression+of+Cre+recombinase+in+hypertrophic+cartilage+under+the+control+of+a+BAC-Col10a1+promoter.">Specific expression of Cre recombinase in hypertrophic cartilage under the control of a BAC-Col10a1 promoter.</a> Matrix Biol. 27 (8), 693-699 (2008).</li><li>Kalajzic, Z., 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=Directing+the+expression+of+a+green+fluorescent+protein+transgene+in+differentiated+osteoblasts:+comparison+between+rat+type+I+collagen+and+rat+osteocalcin+promoters.">Directing the expression of a green fluorescent protein transgene in differentiated osteoblasts: comparison between rat type I collagen and rat osteocalcin promoters.</a> Bone. 31 (6), 654-660 (2002).</li><li>Akiyama, 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=Osteo-chondroprogenitor+cells+are+derived+from+Sox9+expressing+precursors.">Osteo-chondroprogenitor cells are derived from Sox9 expressing precursors.</a> Proc Natl Acad Sci U S A. 102 (41), 14665-14670 (2005).</li><li>Henry, S. 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=Generation+of+aggrecan-CreERT2+knockin+mice+for+inducible+Cre+activity+in+adult+cartilage.">Generation of aggrecan-CreERT2 knockin mice for inducible Cre activity in adult cartilage.</a> Genesis. 47 (12), 805-814 (2009).</li><li>Ren, Y., Lin, S., Jing, Y., Dechow, P. C., Feng, J. Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+way+to+statistically+analyze+morphologic+changes+in+Dmp1-null+osteocytes.">A novel way to statistically analyze morphologic changes in Dmp1-null osteocytes.</a> Connect Tissue Res. 55, 129-133 (2014).</li><li>Pest, M. A., Beier, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Developmental+biology:+Is+there+such+a+thing+as+a+cartilage-specific+knockout+mouse?.">Developmental biology: Is there such a thing as a cartilage-specific knockout mouse?.</a> Nat Rev Rheumatol. 10 (12), 702-704 (2014).</li><li>Tsang, K. Y., Chan, D., Cheah, K. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fate+of+growth+plate+hypertrophic+chondrocytes:+death+or+lineage+extension?.">Fate of growth plate hypertrophic chondrocytes: death or lineage extension?.</a> Dev Growth Differ. 57 (2), 179-192 (2015).</li><li>Sugimoto, Y., Takimoto, A., Hiraki, Y., Shukunami, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+and+characterization+of+ScxCre+transgenic+mice.">Generation and characterization of ScxCre transgenic mice.</a> Genesis. 51 (4), 275-283 (2013).</li><li>Feng, J. Q., 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=Generation+of+a+conditional+null+allele+for+Dmp1+in+mouse.">Generation of a conditional null allele for Dmp1 in mouse.</a> Genesis. 46 (2), 87-91 (2008).</li><li>Lu, Y., Xie, Y., Zhang, S., Dusevich, V., Bonewald, L. F., Feng, J. Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DMP1-targeted+Cre+expression+in+odontoblasts+and+osteocytes.">DMP1-targeted Cre expression in odontoblasts and osteocytes.</a> J Dent Res. 86 (4), 320-325 (2007).</li><li>Rios, A. C., Fu, N. Y., Lindeman, G. J., Visvader, J. 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+situ+identification+of+bipotent+stem+cells+in+the+mammary+gland.">In situ identification of bipotent stem cells in the mammary gland.</a> Nature. 506 (7488), 322-327 (2014).</li><li>Blanpain, C., Simons, B. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Unravelling+stem+cell+dynamics+by+lineage+tracing.">Unravelling stem cell dynamics by lineage tracing.</a> Nat Rev Mol Cell Biol. 14 (8), 489-502 (2013).</li><li>Huh, W. J., Khurana, S. S., Geahlen, J. H., Kohli, K., Waller, R. A., Mills, 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=Tamoxifen+induces+rapid,+reversible+atrophy,+and+metaplasia+in+mouse+stomach.">Tamoxifen induces rapid, reversible atrophy, and metaplasia in mouse stomach.</a> Gastroenterology. 142 (1), 21-24 (2012).</li><li>Lee, M. H., Kim, J. W., Kim, J. H., Kang, K. S., Kong, G., Lee, M. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+expression+profiling+of+murine+hepatic+steatosis+induced+by+tamoxifen.">Gene expression profiling of murine hepatic steatosis induced by tamoxifen.</a> Toxicol Lett. 199 (3), 416-424 (2010).</li><li>Nakamura, E., Nguyen, M. T., Mackem, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Kinetics+of+tamoxifen-regulated+Cre+activity+in+mice+using+a+cartilage-specific+CreER(T)+to+assay+temporal+activity+windows+along+the+proximodistal+limb+skeleton.">Kinetics of tamoxifen-regulated Cre activity in mice using a cartilage-specific CreER(T) to assay temporal activity windows along the proximodistal limb skeleton.</a> Dev Dyn. 235 (9), 2603-2612 (2006).</li></ol>

På grund af teknologiske begrænsninger, er det altid vanskeligt at undersøge opførslen af celler in vivo. Imidlertid er cellelinie opsporing teknik viser sig at være et effektivt værktøj til at studere cellebiologi 7-9. I denne undersøgelse, vi yderligere forbedre denne protokol ved at kombinere det med immunofluorescens. På denne måde kan celleskæbne defineres af flere beslægtede markører, som udvider anvendelsen af ​​afstamning sporing. Desuden dette samarbejde lokalisering af immu...

Under udvikling, endochondral knogledannelse tegner sig for over 80% af skelet volumen. Det er en udbredt opfattelse, at den begynder med apoptose af hypertrofe chondrocytter. Efterfølgende cellerne fra den underliggende knoglemarv invadere og initiere angiogenese efterfulgt af ny knogle aflejring af knogle marrow- og periosteum-afledte celler 1,2. Cellen skæbne hypertrofe chondrocytter (HC) har imidlertid været et problem for debat i årtier 3. Først blev HC&#39;er betragtet som enden af ​​chondrocyt differentieringsvej, og apoptose blev generelt anset for at være den endelige skæbne HC&#39;er. Nu, nogle forskere antyder, at nogle HC&#39;er mindste kunne overleve og bidrage til endochondral knogledannelse. Selvom de foreslog, at væksten plade chondrocytter havde evnen til at transdifferentiate til osteoblaster baseret på ultrastruktur, immunhistokemisk farvning og in vitro-undersøgelser 46, ingen af disse metoder were endelig demonstrere chondrocyt bidrag til osteoblast afstamning. Cellen afstamning sporing teknik giver en mere stringent måde at studere celle skæbne. Kort fortalt set en rekombinase enzym, som kun udtrykkes i en bestemt type celle, stimulerer ekspressionen af ​​reportergenet. På denne måde er denne celletype og deres efterkommere permanent mærket 7. Cre-loxP-systemet er almindeligt anvendt i afstamning sporing. Cre (rekombinase enzym) vil udskære STOP-sekvensen mellem de to loxP-steder og aktivere reporter i et specifikt cellelinje (figur 1A). I nogle tilfælde kan investigator vælge et gunstigt tidspunkt til at aktivere Cre ved anvendelse af et lægemiddel, såsom tamoxifen, forårsager Cre at sammensmelte til en modificeret form af østrogenreceptoren (Cre ERT2) 8. Fluorescerende journalister er blevet standard i afstamning sporing eksperimenter, fordi de dramatisk reducere kompleksitetenog forbedre nøjagtigheden og effektiviteten af celle skæbne sporing 8,9. tdTomato bliver det bedste valg blandt fluorescerende reportere, da det har den klareste fluorescerende protein og den stærkeste epifluorescens, hvilket gør det nemt visualiseres 7 (figur 1A). Som bruger Rosa26 tdTomato afstamning sporingssystem, har vores gruppe og andre forskere vist, at HC kan ændre deres fænotype i knogleceller under udvikling 10-14. For at opnå dette, har vi udviklet to sæt sporing kombinationer med Rosa26 tdtomato (allestedsnærværende ekspression i alle celler) / Cre (specifikt for chondrocytter) mus: 2.3Col1a1-GFP (specifik for osteoblaster) og immunofluorescens (specifikt for knogleceller). Dataene viser, at begge metoder er levedygtige måder at studere celle skæbne 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

Den cellelinie sporingssystem er blevet anvendt, overvejende udviklingsmæssige biologiske undersøgelser. Anvendelsen af ​​Cre-rekombinase giver mulighed for aktivering af reporter i et specifikt cellelinie og alt afkom. Her brugte vi cellen afstamning sporing teknik til at vise, at chondrocytter direkte omdanne til osteoblaster og osteocytter under lange knogler og mandibular condylus udvikling ved hjælp af to slags Cre, Col10a1-Cre og aggrecan-Cre ERT2 (Agg-Cre ERT2), krydset med Rosa26 tdTomato. Både Col10 og aggrecan er velkendt markører for chondrocytter. På dette grundlag har vi udviklet en ny metode-celle afstamning opsporing sammenholdt med fluorescerende immunhistokemi-at definere celle skæbne ved at analysere ekspressionen af ​​specifikke cellemarkører. Runx2 (en markør for debuterende osteogene celler) og Dentin matrix protein1 (DMP1, en markør for sen-fase osteogene celler) varanvendes til at identificere chondrocyt-afledte knogleceller og deres differentiering status. Denne kombination ikke blot udvider anvendelsen af ​​cellelinie opsporing, men også forenkler genereringen af ​​sammensatte mus. Endnu vigtigere er de tal, placering, og differentiering status for forældre celle afkom vises samtidig, som giver mere information end celle afstamning sporing alene. Afslutningsvis co-anvendelse af celle afstamning sporing teknikker og immunofluorescens er et kraftfuldt værktøj til at undersøge cellebiologi in vivo.

Chondrocytter direkte Transform i knogleceller (osteoblaster og osteocytter) i Mandibular condyl og Long Bone. Aggrecan, en kritisk gen for chondrogenese hovedsageligt udtrykkes i tidlige og modne chondrocytter 18. Som følge heraf injektionen af tamoxifen på 2 uger gamle i Agg-Cre ERT2; Rosa26 tdTomato mus aktiveret røde tomat reporter i alle chond...

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 udviklede to sæt sporing kombinationer i Rosa26 tdtomato (ubikvitært udtrykt i alle celler) / Cre (specifikt udtrykkes i chondrocytter) mus: en med 2.3Col1a1-GFP (specifik for osteoblaster) og en med immunofluorescens (specifikt for knogleceller). Dataene viser den direkte omdannelse af chondrocytter i knogleceller.

Co-lokalisering af Cell Lineage Markers 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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This protocol presents a method to perform quantitative, single-cell in situ analyses of protein expression to study lineage specification in mouse ...

Cell Lineage Analyses and Gene Function Studies Using Twin-spot MARCM

Cell Lineage Analyser og genfunktion undersøgelser under anvendelse af 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 af protein-proteininteraktioner og co-lokalisering mellem komponenter af gap-, stram- og adherensforbindelser i murine mammakirtler

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 af humaniserede knoglemarv mikro miljøer i mus og deres visualisering ved 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

Metoder til billeddannelse intracellulære pH af follikel stamcelle afstamning i Live Drosophila æggestokkene væv

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 afstamning omprogrammering af voksne mus Fibroblast til Erythroid ophav

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

Isotropisk lys-Sheet mikroskopi og automatiseret celle Lineage analyser til kataloget Caenorhabditis elegans Embryogenesis med Subcellular opløsning

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