This study was supported by NIH grant DE025014 to JQF.

Tutti i protocolli sono stati esaminati e approvati dal Comitato Istituzionale Animal Care e Usa (IACUC) presso la Texas A &amp; M University College of Dentistry. Allevamento 1. Animal <ol><li> Utilizzare tre modelli animali in questo studio. Per studiare il destino dei condrociti embrionali in formazione condilo, primo utilizzo Col10a1-Cre 15 topi ed attraversarle con Rosa26 tdTomato (B6; 129S6- Gt (ROSA) 26Sor TM9 (CAG-tdTomato) HZE / J) topi per ottenere Col10a1- topi Cre e Rosa26 tdTomato. Successivamente, attraversare questi topi con topi 2.3Col1a1-GFP 16. Utilizzare Col10a1-Cre; Rosa26 tdTomato; Topi 2.3Col1a1-GFP per eseguire gli esperimenti linea cellulare di tracciamento (Figura 1B). </li><li> Per studiare il destino dei condrociti postnatali, seguire la stessa procedura al punto 1.1, ma utilizzare il ERT2 Aggrecan-cre (Agg-Cre ERT2) 17,18 line e mantenere la Agg-Cre ERT2; Rosa26 tdTomato; Topi 2.3Col1a1-GFP per eseguire gli esperimenti linea cellulare Tracing (Figura 1C). Iniettare tamoxifene su postnatale giorno 14. </li><li> Per combinare la linea cellulare tecnica con immunofluorescenza rintracciamento, attraversare topi Agg-Cre ERT2 e topi Rosa26 tdTomato. Utilizzare i topi che portano entrambi i geni nell&#39;esperimento e iniettare il tamoxifene in postnatale giorno 3 (Figura 1D). </li></ol> 2. Preparazione Materiale <ol><li> Sciogliere la polvere tamoxifen in 10% di etanolo e olio di mais al 90% ad una concentrazione di 10 mg / ml. Il dosaggio iniettabile è 75 mg / kg. </li><li> Sciogliere la polvere paraformaldeide (PFA) in PBS ad una concentrazione di 4%, utilizzando 2 M di sodio idrato per regolare il pH a 7,4. Utilizzare 4% soluzione PFA per riparare il tessuto (il condilo mandibolare e gamba posteriore sono utilizzati qui) dopo il sacrificio. A causa del suotossicità, maniglia PFA nella cappa con guanti e una maschera facciale. </li><li> Sciogliere la polvere EDTA in acqua distillata ad una concentrazione del 10%, utilizzando 2 M di sodio idrato per regolare il pH a 7,4. Utilizzare il 10% EDTA per decalcificazione del condilo mandibolare e gamba posteriore. </li><li> Sciogliere in polvere di saccarosio in PBS a concentrazioni di 15% e 30%. Utilizzare la soluzione di saccarosio per disidratare i tessuti dopo la decalcificazione. </li><li> Sciogliere la polvere ialuronidasi in PBS ad una concentrazione di 2 mg / ml, pH 5,0. Questa soluzione è per il recupero immunofluorescenza antigene. </li><li> Utilizzare una provetta da 1,5 ml per preparare una soluzione di saturazione contenente 3% di albumina sierica bovina (BSA) e 20% siero di capra in PBS per Runx2 o DMP1 immunofluorescenza. </li><li> Utilizzare una provetta da 1,5 ml per preparare una soluzione anticorpo primario che contiene 2% di siero di capra in PBS per Runx2 o DMP1 immunofluorescenza colorazione. La concentrazione dell&#39;anticorpo primario è 1: 400 per Runx2 e 1: 100 per DMP1. </li><li> Utilizzare un tubo da 1,5 ml per prepararela soluzione rabbit IgG come controllo per immunofluorescenza per evitare falsi risultati positivi. La soluzione contiene 2% siero di capra in PBS. La concentrazione di IgG di coniglio per il controllo Runx2 è 1: 400; per DMP1, 1: 100. Eseguire l&#39;esperimento e di controllo colorazione contemporaneamente. </li><li> Utilizzare una provetta da 1,5 ml per preparare una soluzione di anticorpo secondario che contiene 2% di siero di capra in PBS per Runx2 o DMP1 immunofluorescenza colorazione. Utilizzare l&#39;anticorpo secondario ad una diluizione di 1: 500. </li></ol> 3. Far scorrere Preparazione per la microscopia confocale (cellulare Lineage Tracing Solo, nessuna combinazione con immunofluorescenza) <ol><li> Iniettare tamoxifene nel Agg-cre ERT2 topi in un punto di tempo favorevole. <ol><li> In primo luogo, rimuovere un mouse dalla sua gabbia. Quindi, utilizzare il pollice e l&#39;indice sinistro per afferrare la pelle del dorso del mouse e capovolgerlo, esponendo l&#39;addome. Usare la mano destra per tenere la siringa. Il punto di ingresso ottimale per l&#39;iniezione è sul lEFT o sul lato destro della all&#39;ipogastrio, evitando il fegato e vescica. </li><li> Tenere la siringa parallelo alle zampe posteriori del mouse e iniettare per via intraperitoneale. Il dosaggio iniettabile è 75 mg / kg. I pesi dei topi all&#39;età di 2 settimane, 3 settimane e 4 settimane di vita sono circa 7-9 g, 11 - 13 g, e 16 - 18 g, rispettivamente. </li></ol></li><li> Anestetizzare i topi con una combinazione Xilazina / Ketaset. <ol><li> Per preparare la soluzione di lavoro, prima diluire la xylazina e Ketaset con acqua distillata alla concentrazione di 1 mg / ml e 5 mg / ml, rispettivamente. Avanti, mescolare il Xilazina e Ketaset in un rapporto 1: 2. Il dosaggio per iniezione è di 30 ml / g. </li><li> Iniettare come al punto 3.1. Confermare l&#39;anestesia pizzicando caviglia del mouse. Il mouse è incosciente se non ha alcuna reazione. </li></ol></li><li> Perfusione i topi con 4% PFA dopo anestesia. <ol><li> Dopo il mouse perde conoscenza, fissare le quattro gambe del mouse su una scheda per entirely esporre l&#39;addome. </li><li> Saturare l&#39;addome con il 70% di etanolo ed effettuare una escissione dal basso addome al collo lungo la linea mediana. Pinch e contemporaneamente tirare la pelle ai fianchi laterali per rivelare la membrana peritoneale. Usare le forbici dissezione per fare un escissione longitudinale. </li><li> Tagliare e rimuovere le costole anteriori per esporre il cuore. Forare il cuore dal ventricolo sinistro con una siringa da 22 G, tenere la siringa, e contemporaneamente tagliare una fessura nel padiglione auricolare destro. </li><li> Iniettare lentamente il 4% PFA, che viene perfuso lungo il sistema cardiovascolare, mentre le vampate sangue dal taglio dal atrio destro. Il volume della PFA per perfusione è 1 ml / grammo. Eseguire questo passaggio in un armadio biosicurezza di classe I che è difficile-canalizzato per il sistema di scarico dell&#39;edificio. </li></ol></li><li> Rimuovere la pelle del topo e si inserisce il corpo intero in una provetta da centrifuga in polipropilene da 50 ml contenente 40 ml di 4% PFA fissare notte a 4 ° C. </li><li>Utilizzare forbici dissezione e # 3 e # 5 pinze per rimuovere accuratamente la mandibola e gamba posteriore dal corpo e per rimuovere i muscoli e tendini sulla superficie. Eseguire questo passaggio in un armadio biosicurezza di classe I che è difficile-canalizzato per il sistema di scarico dell&#39;edificio. </li><li> Tagliare la mandibola in due parti in corrispondenza della zona distale del terzo molare. Analogamente, tagliare il femore e la tibia in mediopeniene per esporre la cavità del midollo osseo al fine di accelerare la decalcificazione. Mettere la parte che comprende il processo condilo e condilare e con la zampa posteriore in 40 ml di 10% EDTA decalcificare a 4 ° C per 2 - 4 giorni in una provetta di polipropilene da centrifuga da 50 ml. </li><li> Utilizzare 50 ml di 15% di saccarosio per disidratare il condilo e zampa posteriore notte a 4 ° C in una provetta di polipropilene da centrifuga da 50 ml. </li><li> Utilizzare 30% di saccarosio per disidratare il condilo e zampa posteriore notte a 4 ° C in un 50-ml tubo di polipropilene centrifuga. </li><li> Lungo il piano sagittale, incorporare il campione conOttobre sul piatto di taglio nella macchina cryosection. <ol><li> Orizzontalmente gettare le condilo o la zampa posteriore nello stampo di montaggio. Immergere il tessuto in OCT e lasciarlo in macchina cryosection fino personalizzazione di Office si blocca. </li><li> Montare il blocco di ottobre sulla piastra di taglio. Attendere circa 15 minuti prima del taglio per garantire che i PTOM è completamente congelato. </li></ol></li><li> Tagliare il condilo e gamba posteriore in sezioni di 10 micron. Raccogliere le sezioni su diapositive e conservare a -20 ° C. </li><li> Incubare il vetrino in una camera 37 ° C per rimuovere l&#39;acqua prima della colorazione. </li><li> Lavare i vetrini due volte con acqua distillata per 5 min. </li><li> Rimuovere l&#39;acqua intorno ogni sezione. Utilizzare una penna barriera idrofobica a fare il giro delle sezioni e rilasciare DAPI o non-fluorescenti soluzione di montaggio anti-sbiadimento nel cerchio. adagiarvi la polizza di copertura. </li></ol> 4. la colorazione immunoistochimica per Runx2 e DMP1 NOTA: L&#39;aria IgGcontrollo è necessario per la colorazione immunoistochimica per evitare segnali falsi-positivi. La colorazione per i gruppi trattati e di controllo deve essere eseguita simultaneamente. <ol><li> Incubare i vetrini in una camera 37 ° C per rimuovere l&#39;acqua prima della colorazione. </li><li> Lavare i vetrini due volte con acqua distillata. </li><li> Utilizzare la penna barriera idrofobica a cerchio tutte le sezioni sul vetrino. Da questo punto, aggiungere tutta la soluzione preparata nel cerchio per coprire completamente le sezioni. </li><li> Trattare le sezioni con ialuronidasi in una camera umida a 37 ° C per 30 min. Il volume della soluzione (a passi di 4,5 - 4,8) dipende dalle dimensioni della sezione. Utilizzare 50 ml di soluzione per il condilo e 100 ml per il lungo osso. Lavare con PBST (PBS contenente 0,1% di Tween 20) tre volte. </li><li> Preparare e applicare la soluzione di blocco per ogni sezione e incubare in una camera umida per 1 ora a temperatura ambiente. </li><li> Incubare le sezioni con primariesoluzione di anticorpi (Runx2 coniglio anti-topo o di coniglio anti-topo DMP1) a 4 ° C durante la notte. Lavare con PBS per tre volte. </li><li> Incubare le sezioni con soluzione di anticorpo secondario (goat anti-rabbit, Alexa Fluor 488) per 2 ore a temperatura ambiente. Lavare con PBS per tre volte. </li><li> Pulire l&#39;acqua intorno alla sezione e rilasciare DAPI nel cerchio per coprire la sezione sulla diapositiva. adagiarvi la polizza di copertura. </li></ol> 5. Microscopia confocale <ol><li> Catturare immagini di cellule fluorescenti usando un microscopio confocale a lunghezze d&#39;onda che vanno da 488 micron (verde) a 561 micron (rosso). Una serie di immagini sovrapposte a 200 Hz (dimensioni di 1.024 × 1.024) 19 utilizzando 10X, 20X, 63X e lenti. </li></ol>

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	<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>

The authors declare that they have no competing financial interests or other conflicts of interest.

A causa di limitazioni tecnologiche, è sempre difficile studiare il comportamento delle cellule in vivo. Tuttavia, la tecnica linea cellulare tracciamento sta dimostrando di essere un potente strumento per lo studio della biologia delle cellule 7-9. In questo studio, abbiamo migliorare ulteriormente questo protocollo attraverso la combinazione con immunofluorescenza. In questo modo, il destino della cellula può essere definita da più marcatori correlate, che estende l&#39;applicazione di lineage t...

Durante lo sviluppo, rappresenta la formazione di osso endochondral per oltre l&#39;80% del volume scheletrico. E &#39;opinione diffusa che inizia con l&#39;apoptosi dei condrociti ipertrofici. Successivamente, le cellule dal midollo osseo sottostante invadono e avviare l&#39;angiogenesi, seguita dalla deposizione di nuovo tessuto osseo da dal midollo osseo e cellule del periostio di derivazione 1,2. Il destino delle cellule di condrociti ipertrofici (HC), tuttavia, è stato un problema di dibattito per decenni 3. Inizialmente, HC sono stati considerati alla fine del percorso di differenziazione dei condrociti, e l&#39;apoptosi è stato generalmente pensato per essere il destino finale di HC. Ora, alcuni ricercatori suggeriscono che almeno alcuni HC potrebbero sopravvivere e contribuire alla formazione di osso endochondral. Anche se hanno proposto che la crescita condrociti piatto aveva la capacità di transdifferenziare in osteoblasti sulla base di ultrastruttura, colorazione immunoistochimica, e studi in vitro 46, nessuno di questi metodi were definitiva a dimostrare il contributo di condrociti alla stirpe degli osteoblasti. La tecnica di linea cellulare tracciare fornisce un modo più rigoroso per studiare il destino della cellula. Per dirla in breve, un enzima ricombinasi, che viene espresso solo in un tipo specifico di cellula, stimola l&#39;espressione del gene reporter. In questo modo, questo tipo di cellula e loro discendenti sono permanentemente etichettati 7. Il sistema Cre-loxP è comunemente usato in lineage tracing. Cre (l&#39;enzima ricombinasi) sarà asportare la sequenza di STOP tra i due siti loxP e attivare il reporter di una determinata linea cellulare (Figura 1A). In alcuni casi, il ricercatore può scegliere un punto tempo favorevole per attivare Cre utilizzando un farmaco, come il tamoxifene, causando Cre di fondere ad una forma modificata del recettore dell&#39;estrogeno (Cre ERT2) 8. giornalisti fluorescenti sono diventati lo standard in lignaggio tracciare esperimenti perché riducono drasticamente la complessitàe migliorare la precisione e l&#39;efficienza del destino delle cellule tracciamento 8,9. tdTomato sta diventando la scelta migliore tra i reporter fluorescenti poiché ha la proteina fluorescente chiaro e il epifluorescenza forte, che lo rende facilmente visualizzato 7 (Figura 1A). Usando il lignaggio Rosa26 tdTomato sistema di rintracciamento, il nostro gruppo e altri ricercatori hanno dimostrato che l&#39;HC può cambiare il loro fenotipo in cellule ossee durante lo sviluppo 10-14. Per fare questo, abbiamo sviluppato due serie di tracciare combinazioni con Rosa26 tdtomato (espressione ubiquitaria in tutte le cellule) / Cre (specifico per condrociti) topo: 2.3Col1a1-GFP (specifico per osteoblasti) e immunofluorescenza (specifico per le cellule ossee). I dati dimostrano che i due metodi sono modi vitali per studiare il destino della cellula in vivo.

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

co-localization of cell lineage markers and the tomato signal

Il sistema di tracciamento linea cellulare è stata utilizzata prevalentemente negli studi di biologia dello sviluppo. L&#39;uso di Cre ricombinasi consente l&#39;attivazione del reporter in una linea cellulare specifica e tutto progenie. Qui, abbiamo utilizzato la linea cellulare tecnica tracing per dimostrare che condrociti trasformano direttamente in osteoblasti e osteociti durante osso lungo e lo sviluppo del condilo mandibolare utilizzando due tipi di Cre, Col10a1-Cre e Aggrecan-Cre ERT2 (Agg-Cre ERT2), incrociati con Rosa26 tdTomato. Sia Col10 e aggrecan sono marcatori ben riconosciuti per condrociti. Su questa base, abbiamo sviluppato un nuovo metodo di lignaggio cellule tracing in combinazione con fluorescenti immunoistochimica-per definire il destino della cellula, analizzando l&#39;espressione di marcatori cellulari specifici. Runx2 (un marker per le cellule osteogeniche fase iniziale) e dentina matrice protein1 (DMP1, un marker per le cellule in fase avanzata osteogeniche) eranoutilizzato per identificare le cellule ossee chondrocyte-derivati ​​e il loro stato di differenziazione. Questa combinazione non solo amplia l&#39;applicazione della linea cellulare tracing, ma semplifica anche la generazione di topi composti. Ancora più importante, gli stati posizione, il numero e la differenziazione delle progenie cellula madre vengono visualizzati contemporaneamente, fornendo più informazioni rispetto linea cellulare tracciamento solo. In conclusione, la co-applicazione di tecniche tracing linea cellulare e immunofluorescenza è un potente strumento per studiare biologia cellulare in vivo.

I condrociti Direttamente trasformarsi in cellule ossee (osteoblasti e osteociti) nel mandibolare Condilo e delle ossa lunghe. Aggrecan, un gene critico per condrogenesi, si esprime soprattutto nelle prime e mature condrociti 18. Come risultato, l&#39;iniezione di tamoxifene a 2 settimane di età in AGG-Cre ERT2; Topi Rosa26 tdTomato attivati il giorn...

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

Abbiamo sviluppato due serie di tracciare combinazioni a Rosa26 tdtomato (ubiquitariamente espresso in tutte le cellule) / Cre (specificamente indicato nella condrociti) topi: uno con 2.3Col1a1-GFP (specifico per osteoblasti) e uno con immunofluorescenza (specifico per le cellule ossee). I dati dimostrano la trasformazione diretta di condrociti in cellule ossee.

Co-localizzazione di marcatori di cellule lignaggio e il segnale di pomodoro

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

co-localization-of-cell-lineage-markers-and-the-tomato-signal

Research

JoVE Journal

Developmental Biology

12.1K Views.  Texas A&M University College of Dentistry.  Abbiamo sviluppato due serie di tracciare combinazioni a Rosa26 tdtomato (ubiquitariamente espresso in tutte le cellule) / Cre (specificamente indicato nella condrociti) topi: uno con 2.3Col1a1-GFP (specifico per osteoblasti) e uno con immunofluorescenza (specifico per le cellule ossee). I dati dimostrano la trasformazione diretta di condrociti in cellule ossee. 

Video: Co-localization of Cell Lineage Markers and the Tomato Signal

A Method for Lineage Tracing of Corneal Cells Using Multi-color Fluorescent Reporter Mice

Lineage tracing experiments define the origin, fate and behavior of cells in a specific tissue or organism. This technique has been successfully applied for many decades, revealing seminal findings in developmental biology. More recently, it was adopted by stem cell biologists to identify and track different stem cell populations with minimal experimental intervention. The recent developments in mouse genetics, the availability of a large number of mouse strains, and the advancements in fluorescent microscopy allow the straightforward design of powerful lineage tracing systems for various tissues with basic expertise, using commercially available tools. We have recently taken advantage of this powerful methodology to explore the origin and fate of stem cells at the ocular surface using R26R-Confetti mouse. This model offers a multi-color genetic system, for the expression of 4 fluorescent genes in a random manner. Here we describe the principles of this methodology and provide an adaptable protocol for designing lineage tracing experiments; specifically for the corneal epithelium as well as for other tissues.

In this article we describe the principles for designing and performing a multi-color lineage tracing experiment using R26R-Confetti mice. We provide a specific protocol for tracking corneal epithelial cells which can be modified for other tissues of interest.

Un metodo per Lineage Tracing di cellule corneali Utilizzando multicolori topi reporter fluorescente

Lineage tracing experiments define the origin, fate and behavior of cells in a specific tissue or organism. This technique has been successfully applied ...

Ricerca

Biologia dello sviluppo

Cell Lineage Analyses and Gene Function Studies Using 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 studies to depict intricate morphologies and to manipulate the function of genes in subsets of neurons within otherwise unmarked and unperturbed organisms. Genetic mosaics generated in the MARCM system are mediated through site-specific recombination between homologous chromosomes within dividing precursor cells to produce both marked (MARCM clones) and unmarked daughter cells during mitosis. An extension of the MARCM method, called twin-spot MARCM (tsMARCM), labels both of the twin cells derived from a common progenitor with two distinct colors. This technique was developed to enable the retrieval of useful information from both hemi-lineages. By comprehensively analyzing different pairs of tsMARCM clones, the tsMARCM system permits high-resolution neural lineage mapping to reveal the exact birth-order of the labeled neurons produced from common progenitor cells. Furthermore, the tsMARCM system also extends gene function studies by permitting the phenotypic analysis of identical neurons of different animals. Here, we describe how to apply the tsMARCM system to facilitate studies of neural development in Drosophila.

Here, we present a protocol for a mosaic labeling technique that permits the visualization of neurons derived from a common progenitor cell in two distinct colors. This facilitates neural lineage analysis with the capability of birth-dating individual neurons and studying gene function in the same neurons of different individuals.

Le analisi delle cellule lignaggio e studi la funzione del gene Impiegando doppi loco MARCM

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

Confocal Live Imaging of Shoot Apical Meristems from Different Plant Species

The shoot apical meristem (SAM) functions as a conserved stem cell reservoir and it generates almost all aboveground tissues during the postembryonic development. The activity and morphology of SAMs determine important agronomic traits, such as shoot architecture, size and number of reproductive organs, and most importantly, grain yield. Here, we provide a detailed protocol for analyzing both the surface morphology and the internal cellular structure of the living SAMs from different species through laser scanning confocal microscope. The whole procedure from the sample preparation to the acquisition of high resolution three-dimensional (3D) images can be accomplished within as short as 20 minutes. We demonstrate that this protocol is highly efficient for studying not only the inflorescence SAMs of the model species but also the vegetative meristems from different crops, providing a simple but powerful tool to study the organization and development of meristems across different plant species.

This protocol presents how to live image and analyze the shoot apical meristems from different plant species using laser scanning confocal microscopy.

Formazione immagine confocal Live dei meristemi apicali riprese da diverse specie di piante

The shoot apical meristem (SAM) functions as a conserved stem cell reservoir and it generates almost all aboveground tissues during the postembryonic ...

Visualization of IL-22-expressing Lymphocytes Using Reporter Mice

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 transgenic reporter mouse model. We chose to visualize interleukin (IL) 22 gene expression because this cytokine has important activities in the intestine, where it contributes to repair tissues damaged by inflammation. Reporter systems offer considerable advantages over other methods of identifying products in vivo. In the case of IL-22, other studies had first isolated cells from tissues and then re-stimulated the cells in vitro. IL-22, which is normally secreted, was trapped inside cells using a drug, and intracellular staining was used to visualize it. This method identifies cells capable of producing IL-22, but it does not determine whether they were doing so in vivo. The reporter design includes inserting a gene for a fluorescent protein (tdTomato) into the IL-22 gene in such a way that the fluorescent protein cannot be secreted and therefore remains trapped inside the producing cells in vivo. Fluorescent producers can then be visualized in tissue sections or by ex vivo analysis through flow cytometry. The actual construction process for the reporter included recombineering a bacterial artificial chromosome that contained the IL-22 gene. This engineered chromosome was then introduced into the mouse genome. Homeostatic IL-22 reporter expression was observed in different mouse tissues, including the spleen, thymus, lymph nodes, Peyer&#39;s patch, and intestine, by flow cytometry analysis. Colitis was induced by T-cell (CD4+CD45RBhigh) transfer, and reporter expression was visualized. Positive T cells were first present in the mesenteric lymph nodes, and then they accumulated inside the lamina propria of the distal small intestine and colon tissues. The strategy using BACs gave good-fidelity reporter expression compared to IL-22 expression, and it is simpler than knock-in procedures.

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.

Visualizzazione di IL-22 che esprimono Linfociti Utilizzando topi Reporter

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 ...

Immunologia e infezioni

Identification of Plasmodesmal Localization Sequences in Proteins In Planta

Plasmodesmata (Pd) are cell-to-cell connections that function as gateways through which small and large molecules are transported between plant cells. Whereas Pd transport of small molecules, such as ions and water, is presumed to occur passively, cell-to-cell transport of biological macromolecules, such proteins, most likely occurs via an active mechanism that involves specific targeting signals on the transported molecule. The scarcity of identified plasmodesmata (Pd) localization signals (PLSs) has severely restricted the understanding of protein-sorting pathways involved in plant cell-to-cell macromolecular transport and communication. From a wealth of plant endogenous and viral proteins known to traffic through Pd, only three PLSs have been reported to date, all of them from endogenous plant proteins. Thus, it is important to develop a reliable and systematic experimental strategy to identify a functional PLS sequence, that is both necessary and sufficient for Pd targeting, directly in the living plant cells. Here, we describe one such strategy using as a paradigm the cell-to-cell movement protein (MP) of the Tobacco mosaic virus (TMV). These experiments, that identified and characterized the first plant viral PLS, can be adapted for discovery of PLS sequences in most Pd-targeted proteins.

Identification of Plasmodesmal Localization Sequences in Proteins In Planta

Plant intercellular connections, the plasmodesmata (Pd), play central roles in plant physiology and plant-virus interactions. Critical to Pd transport are sorting signals that direct proteins to Pd. However, our knowledge about these sequences is still in its infancy. We describe a strategy to identify Pd localization signals in Pd-targeted proteins.

Identificazione di sequenze di localizzazione Plasmodesmal in proteine In Planta

Plasmodesmata (Pd) are cell-to-cell connections that function as gateways through which small and large molecules are transported between plant cells. ...

In Situ Hybridization for the Precise Localization of Transcripts in Plants

With the advances in genomics research of the past decade, plant biology has seen numerous studies presenting large-scale quantitative analyses of gene expression. Microarray and next generation sequencing approaches are being used to investigate developmental, physiological and stress response processes, dissect epigenetic and small RNA pathways, and build large gene regulatory networks1-3. While these techniques facilitate the simultaneous analysis of large gene sets, they typically provide a very limited spatiotemporal resolution of gene expression changes. This limitation can be partially overcome by using either profiling method in conjunction with lasermicrodissection or fluorescence-activated cell sorting4-7. However, to fully understand the biological role of a gene, knowledge of its spatiotemporal pattern of expression at a cellular resolution is essential. Particularly, when studying development or the effects of environmental stimuli and mutants can the detailed analysis of a gene's expression pattern become essential. For instance, subtle quantitative differences in the expression levels of key regulatory genes can lead to dramatic phenotypes when associated with the loss or gain of expression in specific cell types. 
Several methods are routinely used for the detailed examination of gene expression patterns. One is through analysis of transgenic reporter lines. Such analysis can, however, become time-consuming when analyzing multiple genes or working in plants recalcitrant to transformation. Moreover, an independent validation to ensure that the transgene expression pattern mimics that of the endogenous gene is typically required. Immunohistochemical protein localization or mRNA in situ hybridization present relatively fast alternatives for the direct visualization of gene expression within cells and tissues. The latter has the distinct advantage that it can be readily used on any gene of interest. In situ hybridization allows detection of target mRNAs in cells by hybridization with a labeled anti-sense RNA probe obtained by in vitro transcription of the gene of interest. 
Here we outline a protocol for the in situ localization of gene expression in plants that is highly sensitivity and specific. It is optimized for use with paraformaldehyde fixed, paraffin-embedded sections, which give excellent preservation of histology, and DIG-labeled probes that are visualized by immuno-detection and alkaline-phosphatase colorimetric reaction. This protocol has been successfully applied to a number of tissues from a wide range of plant species, and can be used to analyze expression of mRNAs as well as small RNAs8-14.

In Situ Hybridization for the Precise Localization of Transcripts in Plants

The in situ hybridization protocol described here allows a direct localization of mRNA and small RNA expression at the cellular level with high sensitivity and specificity. The procedure is optimized for paraffin-embedded plant tissue sections, is applicable to a wide range of plants and tissues, and can be completed within ten days.

Ibridazione in situ per la localizzazione precisa delle Trascrizioni nelle piante

With the advances in genomics research of the past decade, plant biology has seen numerous studies presenting large-scale quantitative analyses of gene ...

Biologia

Cell Specific Analysis of Arabidopsis Leaves Using Fluorescence Activated Cell Sorting

After initiation of the leaf primordium, biomass accumulation is controlled mainly by cell proliferation and expansion in the leaves1. However, the Arabidopsis leaf is a complex organ made up of many different cell types and several structures. At the same time, the growing leaf contains cells at different stages of development, with the cells furthest from the petiole being the first to stop expanding and undergo senescence1. Different cells within the leaf are therefore dividing, elongating or differentiating; active, stressed or dead; and/or responding to stimuli in sub-sets of their cellular type at any one time. This makes genomic study of the leaf challenging: for example when analyzing expression data from whole leaves, signals from genetic networks operating in distinct cellular response zones or cell types will be confounded, resulting in an inaccurate profile being generated.
To address this, several methods have been described which enable studies of cell specific gene expression. These include laser-capture microdissection (LCM)2 or GFP expressing plants used for protoplast generation and subsequent fluorescence activated cell sorting (FACS)3,4, the recently described INTACT system for nuclear precipitation5 and immunoprecipitation of polysomes6.
FACS has been successfully used for a number of studies, including showing that the cell identity and distance from the root tip had a significant effect on the expression profiles of a large number of genes3,7. FACS of GFP lines have also been used to demonstrate cell-specific transcriptional regulation during root nitrogen responses and lateral root development8, salt stress9 auxin distribution in the root10 and to create a gene expression map of the Arabidopsis shoot apical meristem11. Although FACS has previously been used to sort Arabidopsis leaf derived protoplasts based on autofluorescence12,13, so far the use of FACS on Arabidopsis lines expressing GFP in the leaves has been very limited4. In the following protocol we describe a method for obtaining Arabidopsis leaf protoplasts that are compatible with FACS while minimizing the impact of the protoplast generation regime. We demonstrate the method using the KC464 Arabidopsis line, which express GFP in the adaxial epidermis14, the KC274 line, which express GFP in the vascular tissue14 and the TP382 Arabidopsis line, which express a double GFP construct linked to a nuclear localization signal in the guard cells (data not shown; Figure 2). We are currently using this method to study both cell-type specific expression during development and stress, as well as heterogeneous cell populations at various stages of senescence.

Cell Specific Analysis of Arabidopsis Leaves Using Fluorescence Activated Cell Sorting

A method for producing Arabidopsis leaf protoplasts that are compatible with fluorescence activated cell sorting (FACS), allowing for studies of specific cell populations. This method is compatible with any Arabidopsis line that expresses GFP in a subset of cells.

Cella di analisi specifica di<em&gt; Arabidopsis</em&gt; Foglie usando fluorescenza activated cell sorting

After initiation of the leaf primordium, biomass accumulation is controlled mainly by cell proliferation and expansion in the leaves1. However, the ...

Lineage Tracing of Inducible Fluorescently-Labeled Stem Cells in the Adult Mouse Brain

A telomerase reverse transcriptase (Tert) lineage-tracing mouse line was developed to investigate the behavior and fate of adult tissue stem cells, by crossing the &#39;Tet-On&#39; system oTet-Cre mouse with a novel reverse tetracycline transactivator (rtTA) transgene linked to the Tert promoter, which we have demonstrated marks a novel population of adult brain stem cells. Here, administration of the tetracycline derivative doxycycline to mTert-rtTA::oTet-Cre mice will indelibly mark a population of cells that express a 4.4 kb fragment of the promoter region of the gene Tert. When combined the Rosa-mTmG reporter, mTert-rtTA::oTet-Cre::Rosa-mTmG mice will express membrane tdTomato (mTomato) until doxycycline treatment induces the replacement of mTomato expression with membrane EGFP (mGFP) in cells that also express Tert. Therefore, when these triple-transgenic lineage tracing mice receive doxycycline (the &#34;pulse&#34; period during which TERT expressing cells are marked), these cells will become indelibly marked mGFP+ cells, which can be tracked for any desirable amount of time after doxycycline removal (the &#34;chase&#34; period), even if Tert expression is subsequently lost. Brains are then perfusion-fixed and processed for immunofluorescence and other downstream applications in order to interpret changes to stem cell activation, proliferation, lineage commitment, migration to various brain niches, and differentiation to mature cell types. Using this system, any rtTA mouse can be mated to oTet-Cre and a Rosa reporter to conduct doxycycline-inducible &#34;pulse-chase&#34; lineage&#160;tracing experiments using markers of stem cells.

The ability to permanently mark stem cells and their progeny with a fluorophore using an inducible transgenic lineage tracing mouse line allows for spatial and temporal analysis of activation, proliferation, migration, and/or differentiation in vivo. Lineage tracing can reveal novel information about lineage commitment, response to intervention(s), and multipotency.

Tracciamento del lignaggio delle cellule staminali fluorescenti inducibili nel cervello del topo adulto

A telomerase reverse transcriptase (Tert) lineage-tracing mouse line was developed to investigate the behavior and fate of adult tissue stem cells, by ...

Neuroscienze

Imaging Spatial Reorganization of a MAPK Signaling Pathway Using the Tobacco Transient Expression System

Visualization of dynamic signaling events in live cells has been a challenge. We expanded an established transient expression system, the biomolecular fluorescent complementation (BiFC) assay in tobacco epidermal cells, from testing protein-protein interaction to monitoring spatial distribution of signal transduction in plant cells. In this protocol, we used the BiFC assay to show that the interaction and the signaling between the Arabidopsis MAPKKK YODA and MAPK6 occur at the plasma membrane. When the scaffold protein BASL was co-expressed, the YODA-MAPK interaction redistributed and spatially co-polarized with CFP-BASL. This modified tobacco expression system allows for quick examination of signaling localization and dynamic changes (less than 4 days) and can accommodate multiple of fluorescent protein colors (at least three). We also presented detailed methods to quantify protein distribution (asymmetric spatial localization, or &#34;polarization&#34;) in tobacco cells. This advanced tobacco expression system has a potential to be used widely for quick testing of dynamic signaling events in live plant cells.

At the subcellular level, signaling events are dynamically modulated by developmental and environmental cues. Here we describe a protocol that employs the tobacco transient expression system to monitor dynamic protein-protein interaction and to disclose spatial organization of signal transduction in plant cells.

Imaging Riorganizzazione spaziale di una via di segnalazione MAPK Utilizzando il sistema transitorio Expression Tabacco

Visualization of dynamic signaling events in live cells has been a challenge. We expanded an established transient expression system, the biomolecular ...

Lineage Tracing and Clonal Analysis in Developing Cerebral Cortex Using Mosaic Analysis with Double Markers (MADM)

Beginning from a limited pool of progenitors, the mammalian cerebral cortex forms highly organized functional neural circuits. However, the underlying cellular and molecular mechanisms regulating lineage transitions of neural stem cells (NSCs) and eventual production of neurons and glia in the developing neuroepithelium remains unclear. Methods to trace NSC division patterns and map the lineage of clonally related cells have advanced dramatically. However, many contemporary lineage tracing techniques suffer from the lack of cellular resolution of progeny cell fate, which is essential for deciphering progenitor cell division patterns. Presented is a protocol using mosaic analysis with double markers (MADM) to perform in vivo clonal analysis. MADM concomitantly manipulates individual progenitor cells and visualizes precise division patterns and lineage progression at unprecedented single cell resolution. MADM-based interchromosomal recombination events during the G2-X phase of mitosis, together with temporally inducible CreERT2, provide exact information on the birth dates of clones and their division patterns. Thus, MADM lineage tracing provides unprecedented qualitative and quantitative optical readouts of the proliferation mode of stem cell progenitors at the single cell level. MADM also allows for examination of the mechanisms and functional requirements of candidate genes in NSC lineage progression. This method is unique in that comparative analysis of control and mutant subclones can be performed in the same tissue environment in vivo. Here, the protocol is described in detail, and experimental paradigms to employ MADM for clonal analysis and lineage tracing in the developing cerebral cortex are demonstrated. Importantly, this protocol can be adapted to perform MADM clonal analysis in any murine stem cell niche, as long as the CreERT2 driver is present.

Lineage Tracing and Clonal Analysis in Developing Cerebral Cortex Using Mosaic Analysis with Double Markers MADM

A protocol to perform lineage tracing and functional genetic analysis of candidate genes at a single cell level using mosaic analysis with double markers (MADM) is presented. MADM clonal analysis provides a quantitative framework to measure the proliferative behavior, cellular output, and lineage relationship of individual progenitors and their daughter cells.

Tracciamento del lignaggio e analisi clonale nello sviluppo della Corteccia cerebrale utilizzando l'analisi mosaica con doppi marcatori (MADM)

Beginning from a limited pool of progenitors, the mammalian cerebral cortex forms highly organized functional neural circuits. However, the underlying ...