This study was supported by NIH grant DE025014 to JQF.

Tüm protokoller gözden ve Teksas A &amp; Diş Hekimliği M University College&#39;da Kurumsal Hayvan Bakım ve Kullanım Komitesi (IACUC) tarafından onaylanmıştır. 1. Hayvan Yetiştiriciliği <ol><li> Bu çalışmada, üç hayvan modelleri kullanarak. İlk kullanım Col10a1-CRE 15 fareler kondil oluşumunda embriyonik kondrositlerin kaderi araştırmak ve Rosa26 tdTomato onları geçmeye (B6; 129S6- Gt (ROSA) 26Sor tm9 (CAG-tdTomato) Hze / J) farelerde Col10a1- elde etmek için Cre ve Rosa26 tdTomato fareler. Daha sonra, 2.3Col1a1-GFP uygulanan farelerden 16 bu fareler çapraz. Col10a1-Cre kullanın; Rosa26 tdTomato; 2.3Col1a1-GFP fareler hücre soyu izleme deneyleri (Şekil 1B) gerçekleştirmek için. </li><li> Doğum sonrası kondrosit kaderini incelemek için, adım 1.1 gibi aynı prosedürü izleyin, ama Agrekanın-CRE ERT2 (Agg-Cre ERT2 kullanmak) 17,18 hattı ve Agg-Cre ERT2 tutmak; Rosa26 tdTomato; 2.3Col1a1-GFP fareler hücre soyu izleme deneyleri (Şekil 1C) gerçekleştirmek için. doğum sonrası 14. günde tamoksifen enjekte edilir. </li><li> Immünofloresan ile teknik izleme hücre soyu birleştirmek için, Agg-Cre ERT2 fareler ve Rosa26 tdTomato fareler çapraz. Deneyde her iki geni taşıyan fareler kullanın ve doğum sonrası 3. günde (Şekil 1D) üzerine tamoksifen enjekte edilir. </li></ol> 2. Malzeme hazırlanması <ol><li> 10 mg / ml&#39;lik bir konsantrasyonda% 10 etanol ve% 90 mısır yağı içinde tamoksifen toz eritilir. Enjeksiyon için bir dozaj, 75 mg / kg&#39;dır. </li><li> 7.4 pH değerini ayarlamak için 2M sodyum hidrat kullanılarak,% 4&#39;lük bir konsantrasyona PBS içinde paraformaldehit (PFA) toz içinde çözülür. Kurban sonra doku (mandibular kondil ve arka bacak burada kullanılan) düzeltmek için% 4 PFA çözüm kullanın. Çünkü onuntoksisite, eldiven ve yüz maskesi ile kaputun içindeki PFA anlaştım. </li><li> 7.4 pH değerini ayarlamak için 2M sodyum hidrat kullanılarak,% 10 bir konsantrasyona damıtılmış su içinde EDTA toz eritilir. Mandibular kondil ve arka bacak kirecini% 10 EDTA kullanın. </li><li> % 15 ve% 30 arasında konsantrasyonlarda PBS içinde sükroz toz eritilir. yumuşatma sonra doku kurutmak için sukroz çözümü kullanın. </li><li> 2 mg / ml, pH 5.0 bir konsantrasyonda PBS içinde hyaluronidaz toz eritilir. Bu çözüm immünofloresan antijen alımı içindir. </li><li> Runx2 ya DMP1ve immünofloresan boyama PBS içinde% 3 sığır serum albümini (BSA) ve% 20 keçi serumu içeren bir bloklama solüsyonu hazırlamak için, 1.5 ml tüp kullanın. </li><li> Runx2 ya DMP1ve immünofloresan boyama PBS içinde% 2 keçi serumu ihtiva eden bir birincil antikor bir çözelti hazırlamak için 1.5 ml tüp kullanın. Runx2 400 ve 1: 100 DMP1ve için primer antikor konsantrasyonu, 1&#39;dir. </li><li> hazırlamak için, 1.5 ml tüp kullanarakYanlış pozitif sonuçlardan kaçınmak için immunofluorescent boyama kontrol olarak tavşan IgG çözüm. Çözelti PBS içinde% 2 keçi serumu içerir. Runx2 kontrolü için tavşan IgG konsantrasyonu 1: 400; 100: DMP1ve, 1. Aynı anda deney ve kontrol boyama yapın. </li><li> Runx2 ya DMP1ve immünofloresan boyama PBS içinde% 2 keçi serumu ihtiva eden bir ikincil antikor çözeltisi hazırlamak için, 1.5 ml tüp kullanın. 500: 1 bir seyreltmede ikincil antikor kullanın. </li></ol> 3. Slayt Konfokal Mikroskopi Hazırlık (Sadece izleme Hücre Lineage, İmmünofloresan ile hiçbir Kombinasyon) <ol><li> Olumlu bir zaman noktasında Agg-CRE ERT2 farelerde tamoksifen enjekte edilir. <ol><li> İlk olarak, kafes fare kaldırın. Ardından, karın açığa farenin arkasındaki deriyi kapmak ve ters çevirin sol başparmak ve işaret parmağınızı kullanın. şırıngayı tutmak için sağ elinizi kullanın. Enjeksiyon için uygun bir giriş noktası L verildieft veya hypogastrium sağ tarafında, karaciğer ve mesane kaçınarak. </li><li> fare arka ayakları şırınga paralel tutun ve intraperitoneal enjekte. Enjeksiyon için bir dozaj, 75 mg / kg&#39;dır. sırasıyla 18 g - 9 g, 11 - - 13 g, 16 2 hafta, 3 hafta, ve eski 4 haftalık yaşlarda faresinin ağırlıkları yaklaşık 7&#39;dir. </li></ol></li><li> Bir Xylazine / Ketaset kombinasyonu ile fareler anestezi. <ol><li> çalışma çözeltisi hazırlamak için, ilk olarak sırası ile, 1 mg / ml ve 5 mg / ml &#39;lik bir konsantrasyona kadar damıtılmış su ile iksilazin ve Ketaset seyreltin. 2 oranında: Sonraki, bir 1 ketamin ve Ketaset karıştırın. Enjeksiyon için bir dozaj 30 ul / g&#39;dır. </li><li> Adım 3.1 olarak enjekte edilir. Farenin bileğini kısma anesthetization onaylayın. o hiçbir tepki varsa fare bilinçsiz olduğunu. </li></ol></li><li> anesthetization sonra% 4 PFA ile fareler serpmek. <ol><li> Fare bilincini kaybeder sonra entirel bir gemide fare dört bacağı düzeltmeky karın maruz kalmaktadır. </li><li> % 70 etanol ile karın doyurmak ve orta hat boyunca boyun alt karın bir eksizyonu yapmak. periton zarı ortaya çıkarmak için yan taraflarına cilt çekin aynı anda çimdik ve. uzunlamasına eksizyon yapmak için diseksiyon makas kullanın. </li><li> kesiniz ve kalp maruz ön kaburga kaldırın. Sağ kulak kepçesi bir yuvayı kesmek aynı zamanda, bir 22 G şırınga ile sol ventrikül kalbi delinme şırıngayı tutun ve. </li><li> Yavaş yavaş kardiyovasküler sistem üzerinde perfüze% 4 PFA, enjekte ederken sağ kulak kepçesi gelen kesme dışında kan basması. perfüzyon için PFA hacmi 1 ml / gramdır. Sert-Kanallı bina egzoz sistemine bir Sınıf I biyogüvenlik kabini içinde bu adımı gerçekleştirin. </li></ol></li><li> fare deri soyulabilir ve 4 ° C&#39;de gece boyunca düzeltmek için% 4 PFA içinde 40 ml içeren 50 ml&#39;lik polipropilen santrifüj tüpü içine bütün vücudu koydu. </li><li>dikkatle vücuttan çene ve arka bacak kaldırmak için diseksiyon makas ve # 3 ve # 5 forseps kullanabilir ve yüzeyde kasları ve tendonları kaldırmak için. Sert-Kanallı bina egzoz sistemine bir Sınıf I biyogüvenlik kabini içinde bu adımı gerçekleştirin. </li><li> Üçüncü molar distal bölgede iki parçaya mandibula kesin. Benzer şekilde, decalcification hızlandırmak amacıyla kemik iliği boşluğuna maruz orta cisim olarak femur ve tibia kesti. kondil ve kondiler işlemi kapsamaktadır parçası koymak ve% 10 EDTA, 40 ml arka bacak ile birlikte 2, 4 ° C&#39;de kirecini göre - 50-ml polipropilen santrifüj tüpüne 4 gün. </li><li> 50 ml polipropilen santrifüj tüpüne 4 ° C&#39;de kondil ve gece boyunca arka bacak kurutmak için% 15 sakroz, 50 ml kullanın. </li><li> 50 ml polipropilen santrifüj tüpü içinde gece boyunca 4 ° C&#39;de kondil ve arka bacak kurutmak için% 30 sakroz kullanın. </li><li> sagital düzlem boyunca, örnek ile gömmekcryosection makinesinde kesici plaka Ekim <ol><li> Yatay kondili veya montaj kalıp içinde arka bacak yatıyordu. OCT doku Batmak ve Ekim donuyor kadar cryosection makinede bırakın. </li><li> Kesme plaka üzerinde Ekim bloğu monte edin. Ekim tamamen donmuş olduğundan emin olmak için kesme işleminden önce yaklaşık 15 dakika boyunca bekleyin. </li></ol></li><li> 10 mikron bölüme kondili ve arka bacak kesti. -20 ° C&#39;de slaytlar ve mağaza bölümleri toplayın. </li><li> boyama önce suyu çıkarmak için 37 ° C&#39;lik bir oda içinde slayt inkübe edin. </li><li> 5 dakika için damıtılmış su ile iki kez slaytlar yıkayın. </li><li> Her bölümde etrafında suyu siliniz. bölümleri daire ve çember içine DAPI veya non-floresan anti-solmaya montaj çözümü düşmesi hidrofobik bir bariyer kalem kullanın. Dikkatle kapak kayma uzandı. </li></ol> Runx2 ve DMP1ve 4. İmmünohistokimyasal Boyama NOT: IgG conimmünohistokimyasal yanlış pozitif sinyaller önlemek için kumanda gereklidir. Deney ve kontrol grupları için boyama aynı anda yapılması gerekmektedir. <ol><li> boyama önce suyu çıkarmak için 37 ° C&#39;lik bir oda içinde slaytlar inkübe edin. </li><li> damıtılmış su ile iki kez slaytlar yıkayın. </li><li> slayt tüm bölümleri daire için hidrofobik bariyer kalem kullanın. Bu adımdan itibaren, tamamen bölümleri kapsayacak şekilde daire içine hazırlanmış çözümün bütün ekleyin. </li><li> 30 dakika boyunca 37 ° C&#39;de nemli bir oda içinde hyalüronidaz bölümleri tedavi edin. (Adım 4.5 - 4.8) çözeltisinin hacmi bölümün boyutuna bağlıdır. Uzun kemik kondilin için çözelti 50 ul ve 100 ul kullanın. (% 0.1 içeren PBS Tween 20) ile PBST ile üç kez yıkanır. </li><li> Hazırlama ve her bölüm için engelleme çözeltisi uygulanır ve oda sıcaklığında 1 saat süreyle nemli bir oda içinde inkübe. </li><li> Primer ile bölümleri inkübegece boyunca 4 ° C &#39;de antikor çözeltisi (tavşan anti-fare Runx2 ya tavşan anti-fare DMP1ve). PBS ile üç kez yıkayın. </li><li> Oda sıcaklığında 2 st için ikincil antikor çözeltisinin (keçi anti-tavşan, Alexa Fluor 488) sahip olan kısımlar inkübe edin. PBS ile üç kez yıkayın. </li><li> bölümünde etrafında suyu siliniz ve slayt bölümü kapsayacak şekilde çember içine DAPI bırakın. Dikkatle kapak kayma uzandı. </li></ol> 5. Konfokal Mikroskop <ol><li> 488 mikron (yeşil) den 561 um (kırmızı) arasında değişen dalga boylarında bir konfokal mikroskop kullanılarak floresan hücre görüntüleri yakalayabilir. 10X, 20X ve 63X lensler kullanarak 19 200 Hz birden yığılmış görüntü almak (1.024 × 1.024 boyutları). </li></ol>

<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>

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

Teknolojik sınırlamalar nedeniyle, in vivo hücre davranışlarını araştırmak için her zaman zordur. Ancak, hücre soyu izleme tekniği hücre biyolojisi 7-9 çalışmak için güçlü bir araç olduğunu gösteriyor. Bu çalışmada, biz daha fazla immünofloresan ile birleştirerek bu protokolü geliştirmek. Bu şekilde, hücre kaderi soyu izleme uygulaması genişletmektedir birden çok ilişkili markörler tarafından tanımlanabilir. Ayrıca, immünofloresan ve domates sinyalinin bu co-l...

gelişimi sırasında, endokondral kemik oluşumu iskelet hacmi% 80&#39;inden sorumludur. Yaygın olarak hipertrofik kondrositlerin apoptozu ile başlar inanılmaktadır. Daha sonra, altta yatan kemik iliği hücreleri istila ve kemik iliği ve periost kökenli hücreler 1,2 yeni kemik birikimi ile takip anjiyogenezi başlatmak. Hipertrofik kondrositlerin hücre kaderi (HC), ancak, yıllardır 3 tartışma konusu olmuştur. Başlangıçta, HCS kondrosit farklılaşması yolunun sonu olarak kabul edildi ve apoptoz genellikle HCS nihai kaderi olduğu düşünülmüştür. Şimdi, bazı araştırmacılar en azından bazı HCS hayatta olabilir ve endokondral kemik oluşumuna katkıda bulunduğunu göstermektedir. O büyümeyi önerdi rağmen plaka kondrositler ultrastrüktürdeki, immünohistokimyasal dayalı osteoblast içine transdifferentiate yeteneği vardı ve in vitro, 46 bu yöntemlerin w hiçbiri çalışmalarıosteoblast soy kondrosit katkı gösteren kesin ere. Hücre soyu izleme tekniği hücre kaderi incelemek için daha titiz bir yol sağlar. Kısaca söylemek gerekirse, sadece spesifik bir hücre türü ifade edilen bir yeniden bağlayıcı enzim, bildirici genin ekspresyonunu uyarır. Bu şekilde, bu hücrenin tipi ve onların soyundan kalıcı 7 etiketlenir. Cre-loxP sistemi yaygın soy izleme kullanılır. Cre (rekombinaz enzim) iki loxP siteleri arasında DUR dizisi tüketim ve belirli bir hücre hattı (Şekil 1A) &#39;de muhabir aktive eder. Bazı durumlarda, araştırmacı, östrojen reseptörü (Cre ERT2) 8 modifiye edilmiş bir formu kaynaşmaya Cre neden tamoksifen gibi bir ilaç ile Cre aktif hale getirmek için uygun bir zaman noktası seçebilir. Floresan gazeteciler onlar dramatik karmaşıklığını azaltmak için deneyler izleme soy standart haline gelmiştirve 8,9 izleme hücre kaderinin doğruluğunu ve verimliliğini artırmak. bunun kolayca 7 (Şekil 1A) görüntülenmiştir yapma, parlak floresan proteini ve güçlü Epifloresans beri tdTomato floresan gazetecilere arasında en iyi seçenek haline geliyor. Sistem izleme Rosa26 tdTomato soy kullanarak, grubumuz ve diğer araştırmacılar HCS gelişimi 10-14 esnasında kemik hücreleri içine fenotip değiştirebilir göstermiştir. 2.3Col1a1-GFP (osteoblastlar özgü) ile bağışıklık fluorışıması (kemik hücrelere özgü): Bunu yapmak için, Rosa26 tdTomato (her yerde ifade, tüm hücrelerde) / Cre (kondrosit özgü) fareler ile kombinasyonlar izleme iki set geliştirdi. Veri iki yolun in vivo hücre kaderine çalışma uygulanabilir yollar olduğunu göstermektedir.

<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

Hücre soyu izleme sistemi gelişim biyolojisi çalışmalarında ağırlıklı olarak kullanılmıştır. Cre rekombinazın kullanımı belirli bir hücre çizgisi ve döl muhabir aktivasyonu için izin verir. Burada, kondrositler doğrudan uzun kemik ve Cre, Col10a1-Cre ve Agrekanın-Cre ERT2 (Agg-Cre ERT2) iki çeşit kullanarak mandibular kondil gelişimi sırasında osteoblast ve osteositleri dönüşme olduğunu göstermek için teknik izleme hücre soyu kullanılan Rosa26 ile geçti tdTomato. Col10 ve Agrekanın Hem kondrositlerin için belirteçleri iyi tanınmaktadır. Bu temelde, belirli hücre belirteçleri ifadesini analiz ederek floresan immünohistokimya-tanımlamak hücre kaderi ile birlikte izleme yeni bir yöntem hücre soyu geliştirdi. Runx2 (erken evre osteojenik hücreler için bir işaret) ve dentin matrisi protein1 (DMP1ve, geç evre osteojenik hücreler için bir işaret) idikondrosit türetilmiş kemik hücreleri ve bunların farklılaşma durumunu tanımlamak için kullanılan. Bu kombinasyon, hücre soyu izleme uygulaması genişletir, ama aynı zamanda bileşik farelerin nesil kolaylaştırır sadece. Daha da önemlisi, ana hücre döl sayısı, yeri ve farklılaşma durumları yalnız izleme hücre soyu daha fazla bilgi sağlayan, aynı anda görüntülenir. Sonuç olarak, hücre soyu takip teknikleri ve immünofloresan birlikte uygulamaya in vivo olarak, hücre biyolojisi araştırmak için güçlü bir araçtır.

Kondrositler Doğrudan mandibular ve Uzun Kemik Kemik Hücrelerinin (Osteobalstlar ve osteositlerde) içine Transform. Agrekan, kondrojenez için kritik bir geni, ağırlıklı olarak, erken ve olgun kondrositler 18 olarak ifade edilir. Bunun bir sonucu olarak, Agg-Cre ERT2 bölgesindeki 2 haftalık tamoksifen enjeksiyonu; Rosa26 tdTomato fareler tüm...

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

Immünofloresan ile (osteoblastlar özgü) 2.3Col1a1-GFP ile diğeri (kemik hücrelere özgü): İki / Cre fareler (özellikle kondrosit olarak ifade edilmiştir) (her yerde her zaman bütün hücrelerde ifade edilmiştir) Rosa26 tdTomato kombinasyonları izleme grupları geliştirdi. veri kemik hücrelerine kondrosit doğrudan dönüşümünü göstermektedir.

Hücre Lineage belirteçlerinin Eş-lokalizasyonu ve Domates Sinyali

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 Görüntüleme.  Texas A&M University College of Dentistry.  Immünofloresan ile (osteoblastlar özgü) 2.3Col1a1-GFP ile diğeri (kemik hücrelere özgü): İki / Cre fareler (özellikle kondrosit olarak ifade edilmiştir) (her yerde her zaman bütün hücrelerde ifade edilmiştir) Rosa26 tdTomato kombinasyonları izleme grupları geliştirdi. veri kemik hücrelerine kondrosit doğrudan dönüşümünü göstermektedir. 

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.

Çok renkli Floresan Reporter Fare kullanma Kornea Hücre Lineage İzleme Bir Yöntem

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

Gelişim Biyolojisi

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.

Çift nokta MARCM kullanılması Hücre Lineage Analizleri ve Gen Fonksiyonu Çalışmaları

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.

Sürgün apikal sürgen farklı bitki türü üzerinden confocal canlı görüntüleme

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.

Raportör fareler kullanılarak IL-22 eksprese eden lenfositlerin Görselleştirme

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 ...

İmmünoloji ve Enfeksiyon

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.

Proteinlerin içinde Planta serilerinde Plasmodesmal yerelleştirme tanımlaması

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.

Bitkilerde Transkriptler Hassas Yerelleştirme Situ Hibridizasyon

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

Biyoloji

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.

Hücre Özgül Analizi<em&gt; Arabidopsis</em&gt; Floresans Aktif Hücre Ayırma kullanma Leaves

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.

Yetişkin Fare Beynindeki İndüklenebilir Floresan Etiketli Kök Hücrelerin Soy İzi

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

Nörobilim

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.

Tütün Geçici İfade Sisteminin Kullanılması bir MAPK sinyal yolu Mekansal Reorganizasyon Görüntüleme

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.

Çift Marker (MADM) ile Mozaik Analizi Kullanarak Serebral Korteks Geliştirilmesinde Soy İzleme ve Klonal Analizi

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