Prof. Weiguo Zou ve S. Kato'ya reaktifler ve fareler için ve Zou laboratuvarı üyelerine yararlı tartışmalar için teşekkür ederiz. Ayrıca Şanghay Dokuzuncu Halk Hastanesi Kraniyofasiyal Anomaliler Için Dijitalleştirilmiş Stomatoloji ve Araştırma Merkezi Laboratuvarı'na yardım için teşekkür ediyoruz. Bu çalışma kısmen Çin Ulusal Doğa Bilimleri Vakfı'nın (NSFC) hibeleriyle desteklendi [81570950,81870740,81800949], Şanghay Zirvesi & Plato Disiplinleri, Şanghay Hassas Tıp Enstitüsü'nden SHIPM-mu fonu, Şanghay Dokuzuncu Halk Hastanesi, Şanghay Jiao Tong Üniversitesi Tıp Fakültesi [JC201809], Şanghay Jiao Tong Üniversitesi Tıp Fakültesi için Üst Düzey İnovasyon Ekibinin Teşvik Projesi , Shanghai JiaoTong üniversitesi Tıp Fakültesi Şanghay Dokuzuncu Halk Hastanesi Disiplinler Arası Araştırma Fonu [JYJC201902]. Ve L.J. Üstün Gençlik Tıbbi Yetenekleri, Şanghay "Tıbbi Yeteneğin Yükselen Yıldızları" Gençlik Geliştirme Programı ve Şanghay Jiaotong Üniversitesi'nden "Chen Xing" projesinin bir bilginidir.

Burada açıklanan hayvanlarla ilgili tüm yöntemler Şanghay Jiaotong Üniversitesi Tıp Fakültesi Kurumsal Hayvan Bakım ve Kullanım Komitesi (IACUC) tarafından onaylandı.
1. Osteoklast spesifik Stat3 silme farelerinin yetiştirilmesi
NOT: Stat3fl/fl fareler ticari olarak elde edilmiştir. Ctsk-Cre fareleri S. Kato tarafından sağlanmıştır (Tokyo Üniversitesi, Tokyo, Japonya12). Fareler, standart koşullarda kurumsal hayvan tesisinde belirli patojen içermeyen (SPF) koşullar altında yetiştirildi ve bakımı yapıldı.
<ol><li>Cinsel olarak olgun bir erkek fareyi aynı yaştaki iki dişi fareyle eşleştirin. 18 gün sonra, yenidoğan için günlük kontrolleri kontrol edin. Hamile dişi fareleri ayırın ve gerekirse yalnız tutun. Dişi fareler eşleştirmeden sonraki 1 ay içinde hamile değilse, erkek fareleri farklı üreme kafesleri arasında değiştirin.</li>
	<li>Stat3fl/fl farelerini Ctsk-Cre farelerine (F0) çapraz. Genotipleme için kuyrukları kırpın ve erkek Stat3fl /+; Ctsk-Cre fareleri, yaklaşık 6 haftalık (F1) olan cinsel olarak olgunlaşana kadar. Aşağıdaki astarları kullanın: Stat3 F-TTGACCTGTCTCCTACAAAAAA; Stat3 R-CCCTAGATTAGGCCAGCACA; Ctsk-cre F-GAACGCACTGATTTCGACCA; Ctsk-cre R-GCTAACCAGCGTTTTCGTTCTTC.</li>
	<li>Çapraz 6 haftalık erkek Stat3fl/+; Dişi Stat3fl / fl fareleri ile Ctsk-Cre fareleri. Genotipleme için kuyrukları kırpın ve erkek Stat3 fl / fl'itutun; Ctsk-Cre fareleri, yaklaşık 6 haftalık (F2) olan cinsel olarak olgunlaşana kadar.</li>
	<li>Çapraz 6 haftalık erkek Stat3 fl/fl; Ctsk-Cre fareler ile dişi Stat3fl/fl fareler (F3). Genotipleme için kuyrukları kırpın ve eski üreme farelerini zamanla daha genç farelerle değiştirin (F3 + N).</li>
</ol>2. Numune toplama
<ol><li>Altı çift 8 haftalık erkek Stat3fl/fl ve Stat3Ctsk çöp eş farelerini karbondioksit boğulması ile ayrı ayrı ötenazi edin. NOT: CO2 akış hızı, kafes hacminin dakikada %30'unu yerinden eder (örneğin, 45 cm x 30 cm x 30 cm kafes için CO2 akış hızı 40 L/dk'dır).</li>
	<li>Fareleri bir destek pozisyonuna yerleştirin. bilateral kalça eklemlerini elle hafifçe yerinden sökün. Cildi distal kaval kemiğinden dikey olarak kesmek için oftalmik makas kullanın ve ardından tüm cildi arka uzuvdan parçalara bölün.</li>
	<li>Arka uzvu ayırmak için sağ kalça ekleminin ve diz ekleminin eklem bağını makasla kesin. Trokanteri ve fibulanın birleştiği yerde kesin ve arka uzvu% 4 paraformaldehit içine daldırın. 3. adım için sağ arka uzuvları saklayın. Kemik iliğini% 4 paraformaldehit ile tamamen daldırmak ve düzeltmek için kemiği her iki ucundan uygun şekilde kesin.</li>
	<li>Sol kalça ekleminin ve diz ekleminin eklem bağını makasla kesin, yumuşak dokuyu hafifçe çıkarın ve kaval kemiğini ve uyluk kemiğini dikkatlice ayırın. Kaval kemiğini ve uyluk kemiğini ayrı ayrı% 75 etanol içine daldır. 4. adım için femora ve adım 6 için kaval kemiği tutun. Trochanter'ı sağlam tuttuğunuzdan emin olun.</li>
</ol>3. Parafin bölümü hazırlığı
<ol><li>Sağ arka uzvu 48 saat boyunca 4 °C'de % 4 paraformaldehitte sabitle.</li>
	<li>Kireçten arındır: Numuneleri 1x PBS ile 10 dakika 3 kez hafifçe yıkayın. Kemikleri bükülene kadar 3 ila 4 hafta boyunca bir ultrasonik kireç çözücü ile örnekleri% 15 EDTA'da (800 mL'de 800 mL ddH2O ve 10x PBS'de 100 mL) kireçten arındırın. Her gün taze kireç çözen sıvı ile değiştirin.</li>
	<li>Numuneleri 1x PBS ile 3x hafifçe yıkayın ve ardından bir gecede 4 °C'de% 75 etanol içine daldırın.</li>
	<li>Susuzluk: İkinci gün, örnekleri her biri iki kez 1 saat boyunca% 95 etanol,% 100 etanol ve ksilen içine sırayla daldırma.</li>
	<li>Örnekleri 30 dakika boyunca 1/2 ksilen 1/2 parafin içine daldırın. Numuneleri gece boyunca 65 °C'de parafin içine daldırın.</li>
	<li>Gömme: Gömme için uygun bir gömme tankı seçin. Kaval kemiğini düzgün bir şekilde altına yerleştirin. Uyluk kemiğini ve kaval kemiğini 90° açıyla yerleştirin. Parafin tamamen soğuduktan ve katılaştıktan sonra gömme tankından çıkarın. Örnekleri numaralayın ve gece boyunca -20 °C'de saklayın.</li>
	<li>Mikrotom kullanarak sürekli olarak 5 μm kalınlığında bölümleri kesin. 20-40 bölüm kesin. Bölümleri 37 °C suya yayın, mikroskop slaytlarına yapıştırın ve gece boyunca 42 °C'de pişirin.</li>
</ol>4. Mikro BT tarama ve analizi
<ol><li>Mikro-CT tarayıcı ile sol femorayı tarayın. Çözünürlük: 10 μm; Gerilim: 70 kV; Akım: 114 μA; Fliter: 0,5 mm Al; Dönme adımı: 0,5°.</li>
	<li>Üreticinin talimatını takiben tarayıcının destekleyici yazılımını kullanarak kortikal kemik ve trabeküler kemiğin 3B görüntülerini yeniden yapılandırın. ROI'ler distal büyüme plakasına yakın toplam 1 mm genişlikte trabeküler kemikte ve femoranın ortasında kortikal kemiğin toplam 1 mm genişliğinde bir bölümündedir.</li>
	<li>Nicel mikro mimarisi parametrelerini hesaplayın: kemik mineral yoğunluğu (BMD), kemik hacmi fraksiyonu (BV/TV), trabeküler kalınlık (Tb.Th.), trabeküler sayı (Tb.N.), trabeküler ayırma (Tb.Sp.) ve kortikal kemik kalınlığı (Ct.Th.).</li>
</ol>5. TUZAK boyama
<ol><li>Parafin bölümlerini 65 °C'de 30 dakika pişirin.</li>
	<li>Dewax: Bölümleri 10 dakika boyunca ksilen içine daldırın. Bu adımı taze ksilen ile 3x gerçekleştirin.</li>
	<li>Rehidrat: Bölümleri sırayla% 100 etanol,% 95 etanol,% 70 etanol ve ddH2O'ya daldırın, her biri iki kez 5 dakika boyunca.</li>
	<li>Üreticinin talimatlarına uyarak TRAP boyama kitini kullanarak boyama sokumini hazırlayın ve 37 °C'ye ısıtın. NOT: TRAP boyama sosu her test öncesinde hemen yeni hazırlanmalıdır.</li>
	<li>Her numuneye 50-100 μL boyama çözeltisi ekleyin ve 37 °C nemli bir odada 20-30 dakika kuluçkaya yatırın. Kırmızı çok fonksiyonlu osteoklastlar görülene kadar her 5 dakikada bir ışık mikroskobu altında osteoklastların boyama durumunu kontrol edin. Reaksiyona ddH2O ile son verin.</li>
	<li>30 s için hematoksilin çözeltisinde karşıtlık. 1 dakika boyunca% 1 amonyak çözeltisinde daldırarak kararlı bir mavi renk oluşturun. Yavaşça akan musluk suyunda durulayın.</li>
	<li>Bölümleri nötr balsam kullanarak kapaklarla monte edin ve gece boyunca kurutun.</li>
	<li>3-5 ilgi alanını mikroskopla yakalayın. Trabeküler çevreyi Resim J ile analiz edin: ' düzçizgi' aracını L1 olarak kullanarak ölçek çubuğunun (Ls) uzunluğunu ölçün, ardından 'parçalı çizgi' aracını L2, fiziksel uzunluk (Lp)= Ls * L2 / L1 olarak kullanarak trabeküler çevrenin uzunluğunu ölçün. Trap pozitif hücrelerin sayısını üçten fazla çekirdekle sayın.</li>
</ol>6. Calcein ve alizarin kırmızı çift etiketleme
<ol><li>Numune hazırlama: Intraperitoneally enjekte 20 mg/kg kalsein (1 mg/mL % 2 NaHCO3 çözeltisi içinde) gün 0, ve 25 mg/kg alizarin kırmızı S (AL, H 2 O'da2mg/mL) 4. 7. günde fareleri kurban edin. Kaval kemiğini dikkatlice ayırın ve 48 saat boyunca 4 °C'de% 4 paraformaldehitte sabitleyin.</li>
	<li>Dehidrat: Sabitlemeden sonra kaval kemiğini 3x'i 1x PBS ile hafifçe yıkayın. Numuneleri sırayla% 95 etanol,% 100 etanol ve ksilen için 5 dakika boyunca ayrı ayrı daldırın.</li>
	<li>Numuneleri 12 saat aseton, 2 saat boyunca 1/2 aseton 1/2 reçine ve bir gecede kurutma fırınında saf reçineye daldırin. NOT: Reçine, üreticinin talimatlarına göre piyasada bulunan reçine (örneğin, Embed 812 Reçine) tarafından hazırlanmıştır.</li>
	<li>Gömme: Uygun bir silika jel gömme tankına saf reçine ekleyin ve kabarcıkları önlemek için numuneleri hafifçe yerleştirin. Reçineyi 60 °C'de 48 saat boyunca kurutma fırınında polimerize edin.</li>
	<li>Numuneleri döner mikrotom ile sürekli olarak 5 μm kalınlığında bölümlere kesin. Numunelerin geri kalanını oda sıcaklığında kurutucu ile saklayın.</li>
	<li>Bölümleri% 75 alkol damlasına cımbızla yapıştırın. Bölümleri nötr balsam kullanarak kapaklarla monte edin. Floresan mikroskop ile kırmızı ve yeşil floresan etiketlemeyi yakalayın.</li>
	<li>İki etiketleme çizgisi (Ir.L.Wi), tek etiketli trabeküler çevre (sL.Pm), çift etiketli trabeküler çevre (dL.Pm) ve toplam trabeküler çevre (Tb.Pm) arasındaki genişliği ölçün. Mineral appozisyon oranını (MAR) ve kemik oluşum oranını (BFR/BS) hesaplayın. MAR = Ir.L.Wi/interval days. BFR/BS= (dL.Pm±sL.Pm/2)*MAR/Tb.Pm*%100.</li>
</ol>

<ol>
	<li>Zaidi, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Skeletal+remodeling+in+health+and+disease.">Skeletal remodeling in health and disease.</a> Nature Medicine. 13 (7), 791-801 (2007).</li><li>Boyle, W. J., Simonet, W. S., Lacey, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Osteoclast+differentiation+and+activation.">Osteoclast differentiation and activation.</a> Nature. 423 (6937), 337-342 (2003).</li><li>Cummings, S. R., Melton, L. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epidemiology+and+outcomes+of+osteoporotic+fractures.">Epidemiology and outcomes of osteoporotic fractures.</a> Lancet. 359 (9319), 1761-1767 (2002).</li><li>Black, D. M., Rosen, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clinical+Practice.+Postmenopausal+Osteoporosis.">Clinical Practice. Postmenopausal Osteoporosis.</a> New England Journal of Medicine. 374 (3), 254-262 (2016).</li><li>Kos, C. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cre/loxP+system+for+generating+tissue-specific+knockout+mouse+models.">Cre/loxP system for generating tissue-specific knockout mouse models.</a> Nutrition reviews. 62 (6), 243-246 (2004).</li><li>Elefteriou, F., Yang, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+mouse+models+for+bone+studies-Strengths+and+limitations.">Genetic mouse models for bone studies-Strengths and limitations.</a> Bone. 49 (6), 1242-1254 (2011).</li><li>Hirano, T., Ishihara, K., Hibi, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Roles+of+STAT3+in+mediating+the+cell+growth,+differentiation+and+survival+signals+relayed+through+the+IL-6+family+of+cytokine+receptors.">Roles of STAT3 in mediating the cell growth, differentiation and survival signals relayed through the IL-6 family of cytokine receptors.</a> Oncogene. 19 (21), 2548-2556 (2000).</li><li>Yu, H., Lee, H., Herrmann, A., Buettner, R., Jove, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Revisiting+STAT3+signalling+in+cancer:+new+and+unexpected+biological+functions.">Revisiting STAT3 signalling in cancer: new and unexpected biological functions.</a> Nature Reviews Cancer. 14 (11), 736-746 (2014).</li><li>Itoh, 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=A+critical+role+for+interleukin-6+family-mediated+Stat3+activation+in+osteoblast+differentiation+and+bone+formation.">A critical role for interleukin-6 family-mediated Stat3 activation in osteoblast differentiation and bone formation.</a> Bone. 39 (3), 505-512 (2006).</li><li>Zhou, 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=Osteoblast/osteocyte-specific+inactivation+of+Stat3+decreases+load-driven+bone+formation+and+accumulates+reactive+oxygen+species.">Osteoblast/osteocyte-specific inactivation of Stat3 decreases load-driven bone formation and accumulates reactive oxygen species.</a> Bone. 49 (3), 404-411 (2011).</li><li>Yang, 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=STAT3+controls+osteoclast+differentiation+and+bone+homeostasis+by+regulating+NFATc1+transcription.">STAT3 controls osteoclast differentiation and bone homeostasis by regulating NFATc1 transcription.</a> Journal of Biological Chemistry. 294 (42), 15395-15407 (2019).</li><li>Nakamura, T., 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=Estrogen+prevents+bone+loss+via+estrogen+receptor+alpha+and+induction+of+Fas+ligand+in+osteoclasts.">Estrogen prevents bone loss via estrogen receptor alpha and induction of Fas ligand in osteoclasts.</a> Cell. 130 (5), 811-823 (2007).</li><li>Kim, H., Kim, M., Im, S. K., Fang, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+Cre-LoxP+system:+general+principles+to+determine+tissue-specific+roles+of+target+genes.">Mouse Cre-LoxP system: general principles to determine tissue-specific roles of target genes.</a> Laboratory animal research. 34 (4), 147-159 (2018).</li><li>Minkin, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bone+acid+phosphatase:+tartrate-resistant+acid+phosphatase+as+a+marker+of+osteoclast+function.">Bone acid phosphatase: tartrate-resistant acid phosphatase as a marker of osteoclast function.</a> Calcified Tissue International. 34 (3), 285-290 (1982).</li><li>Vaaraniemi, 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=Intracellular+machinery+for+matrix+degradation+in+bone-resorbing+osteoclasts.">Intracellular machinery for matrix degradation in bone-resorbing osteoclasts.</a> Journal of Bone and Mineral Research. 19 (9), 1432-1440 (2004).</li><li>Ljusberg, 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=Proteolytic+excision+of+a+repressive+loop+domain+in+tartrate-resistant+acid+phosphatase+by+cathepsin+K+in+osteoclasts.">Proteolytic excision of a repressive loop domain in tartrate-resistant acid phosphatase by cathepsin K in osteoclasts.</a> The Journal of Biological Chemistry. 280 (31), 28370-28381 (2005).</li><li>Janckila, A. J., Takahashi, K., Sun, S. Z., Yam, L. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Naphthol-ASBI+phosphate+as+a+preferred+substrate+for+tartrate-resistant+acid+phosphatase+isoform+5b.">Naphthol-ASBI phosphate as a preferred substrate for tartrate-resistant acid phosphatase isoform 5b.</a> Journal of Bone and Mineral Research. 16 (4), 788-793 (2001).</li><li>Janckila, A. J., Li, C. Y., Lam, K. W., Yam, L. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+cytochemistry+of+tartrate-resistant+acid+phosphatase.+Technical+considerations.">The cytochemistry of tartrate-resistant acid phosphatase. Technical considerations.</a> American Journal of Clinical Pathology. 70 (1), 45-55 (1978).</li><li>Janckila, A. J., Simons, R. M., Yam, L. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alternative+immunoassay+for+tartrate-resistant+acid+phosphatase+isoform+5b+using+the+fluorogenic+substrate+naphthol+ASBI-phosphate+and+heparin.">Alternative immunoassay for tartrate-resistant acid phosphatase isoform 5b using the fluorogenic substrate naphthol ASBI-phosphate and heparin.</a> Clinica Chimica Acta: International Journal of Clinical Chemistry. 347 (1-2), 157-167 (2004).</li><li>Janckila, A. J., Yam, L. T., Li, C. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunoalkaline+phosphatase+cytochemistry.+Technical+considerations+of+endogenous+phosphatase+activity.">Immunoalkaline phosphatase cytochemistry. Technical considerations of endogenous phosphatase activity.</a> American Journal of Clinical Pathology. 84 (4), 476-480 (1985).</li><li>Solberg, L. B., 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=Increased+tartrate-resistant+Acid+phosphatase+expression+in+osteoblasts+and+osteocytes+in+experimental+osteoporosis+in+rats.">Increased tartrate-resistant Acid phosphatase expression in osteoblasts and osteocytes in experimental osteoporosis in rats.</a> Calcified Tissue International. 94 (5), 510-521 (2014).</li><li>Tambutt&#233;, E., 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=Calcein+labelling+and+electrophysiology:+insights+on+coral+tissue+permeability+and+calcification.">Calcein labelling and electrophysiology: insights on coral tissue permeability and calcification.</a> Proceedings. Biological Sciences. 279 (1726), 19-27 (2012).</li><li>Han, 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=Lkb1+deletion+in+periosteal+mesenchymal+progenitors+induces+osteogenic+tumors+through+mTORC1+activation.">Lkb1 deletion in periosteal mesenchymal progenitors induces osteogenic tumors through mTORC1 activation.</a> Journal of Clinical Investigation. 130 (5), 1895-1909 (2019).</li><li>Dai, 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=mTOR/Raptor+signaling+is+critical+for+skeletogenesis+in+mice+through+the+regulation+of+Runx2+expression.">mTOR/Raptor signaling is critical for skeletogenesis in mice through the regulation of Runx2 expression.</a> Cell Death and Differentiation. 24 (11), 1886-1899 (2017).</li><li>Sun, 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=Histone+demethylase+LSD1+regulates+bone+mass+by+controlling+WNT7B+and+BMP2+signaling+in+osteoblasts.">Histone demethylase LSD1 regulates bone mass by controlling WNT7B and BMP2 signaling in osteoblasts.</a> Bone Research. 6, 14 (2018).</li></ol>

Yazarların açıklayacak bir şeyi yok.

Genetik olarak tasarlanmış fare modelleri genellikle insan hastalığının mekanizmasını ve farmasötik tedavisini incelemek için kullanılır13. Ctsk-Cre fareler osteoklastların fonksiyonel çalışmaları için yaygın olarak kullanılmaktadır6. Bu çalışmada iskelet fenotipini analiz etmek ve in vivo osteoklast aktivitesini kontrol eden kritik genleri incelemek için yöntemlerin protokolleri açıklanmıştır.
Histolojik a...

İskelet kemiği insan vücudunun ana yük taşıyan organıdır ve yürüyüş ve egzersiz sırasında hem iç hem de dış ortamdan baskı altındadır1. Kişinin yaşamı boyunca, kemikler sürekli olarak osteoblastlar ve osteoklastlar tarafından dengelenen kendini yenilemeden geçer. Osteoklastların eski kemikleri temizleme ve osteoblastların yeni kemik oluşturma süreci, iskelet sisteminin homeostazını ve mekanik işlevini korur2. Dengedeki bozukluk osteoporoz gibi kemik metabolik hastalıklarına neden olabilir. Aşırı osteoklastik aktivitenin neden olduğu osteoporoz, küresel olarak yaygındır ve topluma önemli ekonomik kayıplara neden olur2,3,4. Osteoporoz tedavisi için mevcut sınırlı sayıda ilada ve yan etki risklerine göre4, osteoklast oluşumu ve aktivitesinin ayrıntılarını ortaya çıkarmak önemlidir.
Monosit/makrofaj hematopoetik soyundan elde edilen osteoklastlar birden fazla çekirdeğe sahiptir (2 ila 50 çekirdek olabilir) ve büyüktür (genellikle çapı 100 μm'den büyüktür)2. Mekanizmaların keşfi ve ilaçların osteoklastik bozukluklar için taranma in vitro osteoklast kültürü ile yaygın olarak geliştirilmiş olmasına rağmen, karmaşık organik reaksiyonlar in vivo kanıtları hedeflenen tedavi için vazgeçilmez kılmaktadır. Fareler ve insanlar arasındaki genetik ve patofizyolojik benzerlikler nedeniyle, genetik olarak tasarlanmış fare modelleri, insan hastalığının mekanizmalarını ve farmasötik tedavilerini incelemek için yaygın olarak kullanılmaktadır in vivo6. Cre-loxP sistemi fare gen düzenlemesi için yaygın olarak kullanılan bir teknolojidir ve araştırmacıların gen fonksiyonlarını doku/hücreye özgü bir şekilde araştırmalarını sağlamıştır5. Cathepsin K (CSTK), kemik kollajen8'ibozabilen osteoklastlar tarafından salgılanan sistein proteazdır. CTSK'nın olgun osteoklastlarda seçici olarak ifade edildiği iyi kabul edilmektedir; Bu nedenle, Ctsk-Cre fareleri osteoklastların fonksiyonel çalışmaları için yararlı bir araç olarak kabul edilir ve6.
Transkripsiyon (STAT) ailesinin sinyal dönüştürücüsü ve aktivatörü klasiktir ve bağışıklık ve kanser ilerlemesi ve gelişiminde oldukça önemlidir7,8. Yedi STAT arasında, STAT3'ün kemik homeostazı9,10ile en alakalı olduğu bildirilmektedir. Çeşitli in vivo çalışmalar, OSTEOBLASTLARDA STAT3'ün spesifik inaktivasyonunun kemik oluşumunu azalttığını bildirmektedir9,10. Bununla birlikte, STAT3'ün osteoklast oluşumuna ve kemik metabolizmasına in vivo katılımına ilişkin sağlam kanıtlar hala sınırlıdır. Son zamanlarda, osteoklast spesifik Stat3 silme fare modeli (Stat3fl / fl; Ctsk-Cre, bundan sonra Stat3Ctskolarak adlandırılır) STAT3 osteoklast farklılaşmasına ve kemik metabolizması11'e katılır. Bu çalışmada, osteoklast spesifik STAT3 delesyonunun kemik homeostazı üzerindeki etkisini incelemek için Stat3Ctsk farelerinin kemik kütlesi, kemik histoporfolojisi ve kemik anabolizması ve katabolizmasındaki değişiklikleri analiz etmek için kullandığımız yöntem ve protokolleri açıklıyoruz.

<table>
	<tbody>
		<tr>
			<td>4% Paraformaldehyde solution</td>
			<td>Sangon biotech Co., Ltd.</td>
			<td>E672002</td>
			<td></td>
		</tr>
		<tr>
			<td>Acetone</td>
			<td>Shanghai Experimental Reagent Co., Ltd.</td>
			<td>80000360</td>
			<td></td>
		</tr>
		<tr>
			<td>Alizarin</td>
			<td>Sigma-Aldrich</td>
			<td>A5533</td>
			<td></td>
		</tr>
		<tr>
			<td>Ammonia solution</td>
			<td>Shanghai Experimental Reagent Co., Ltd.</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Calcein</td>
			<td>Sigma-Aldrich</td>
			<td>C0875</td>
			<td></td>
		</tr>
		<tr>
			<td>Ctsk-Cre mice</td>
			<td></td>
			<td></td>
			<td>a gift from S. Kato, University of Tokyo, Tokyo, Japan</td>
		</tr>
		<tr>
			<td>DDSA</td>
			<td>Electron Microscopy Sciences</td>
			<td>13710</td>
			<td></td>
		</tr>
		<tr>
			<td>DeCa RapidlyDecalcifier</td>
			<td>Pro-Cure</td>
			<td>DX1100</td>
			<td></td>
		</tr>
		<tr>
			<td>DMP-30</td>
			<td>Electron Microscopy Sciences</td>
			<td>13600</td>
			<td></td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>Shanghai Experimental Reagent Co., Ltd.</td>
			<td>60-00-4</td>
			<td></td>
		</tr>
		<tr>
			<td>EMBED 812 RESIN</td>
			<td>Electron Microscopy Sciences</td>
			<td>14900</td>
			<td></td>
		</tr>
		<tr>
			<td>fluorescence microscope</td>
			<td>Olympus</td>
			<td>IX73</td>
			<td></td>
		</tr>
		<tr>
			<td>Hematoxylin solution</td>
			<td>Beyotime Biotechanology</td>
			<td>C0107</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro-CT</td>
			<td>Scanco Medical AG</td>
			<td>&#956;CT 80</td>
			<td></td>
		</tr>
		<tr>
			<td>NaHCO3</td>
			<td>Shanghai Experimental Reagent Co., Ltd.</td>
			<td>10018918</td>
			<td></td>
		</tr>
		<tr>
			<td>Neutral balsam</td>
			<td>Sangon biotech Co., Ltd.</td>
			<td>E675007</td>
			<td></td>
		</tr>
		<tr>
			<td>NMA</td>
			<td>Electron Microscopy Sciences</td>
			<td>19000</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraffin</td>
			<td>Sangon biotech Co., Ltd.</td>
			<td>A601889</td>
			<td></td>
		</tr>
		<tr>
			<td>rotary microtome</td>
			<td>Leica</td>
			<td>RM2265</td>
			<td></td>
		</tr>
		<tr>
			<td>Stat3fl/fl mice</td>
			<td>GemPharmatech Co., Ltd</td>
			<td>D000527</td>
			<td></td>
		</tr>
		<tr>
			<td>TRAP staining kit</td>
			<td>Sigma-Aldrich</td>
			<td>387A</td>
			<td></td>
		</tr>
		<tr>
			<td>xylene</td>
			<td>Shanghai Experimental Reagent Co., Ltd.</td>
			<td>1330-20-7</td>
			<td></td>
		</tr>
	</tbody>
</table>

skeletal phenotype analysis of a conditional stat3 deletion mouse model

Transgenik fare modelleri, osteoklast farklılaşmasını ve aktivitesini kontrol eden kritik genleri anlamak ve osteoporozun mekanizmalarını ve farmasötik tedavilerini incelemek için güçlüdür. Cathepsin K (Ctsk)-Cre fareleri osteoklastların fonksiyonel çalışmaları için yaygın olarak kullanılmaktadır. Transkripsiyon 3'ün (STAT3) sinyal dönüştürücüsü ve aktivatörü kemik homeostazında geçerlidir, ancak in vivo osteoklastlardaki rolü zayıf tanımlanmıştır. STAT3'ün osteoklast farklılaşmasına ve kemik metabolizmasına katıldığına dair in vivo kanıtları sağlamak için osteoklast spesifik bir Stat3 silme fare modeli(Stat3 fl/fl; Ctsk-Cre) ve iskelet fenotipini analiz etti. Mikro-CT tarama ve 3D rekonstrüksiyon, koşullu nakavt farelerinde kemik kütlesinin arttığını ima etti. Kemik metabolizmasını tespit etmek için H&E lekelenmesi, kalsesin ve alizarin kırmızı çift boyama ve tarta dayanıklı asit fosfataz (TRAP) lekeleme yapıldı. Kısacası, bu protokol iskelet fenotipini analiz etmek ve in vivo osteoklast aktivitesini kontrol eden kritik genleri incelemek için bazı kurallı yöntem ve teknikleri açıklar.

Mevcut protokol kullanılarak, STAT3 silmenin osteoklast farklılaşması üzerindeki etkisini incelemek için osteoklast spesifik Stat3 silme fareleri üretildi. Stat3Ctsk fareleri ve wildtype (WT) çöp arkadaşları genotiplemeden sonra yetiştirildi ve tutuldu. Kemik iliği makrofajları izole edildi ve osteoklastlar halinde kültürlendi ve Stat3Ctsk farelerinde STAT3delesyonu gösterildi (Şekil 1).
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Watch this Scientific Journal Video about Skeletal Phenotype Analysis of a Conditional Stat3 Deletion Mouse Model at JoVE.com

Skeletal Phenotype Analysis of a Conditional Stat3 Deletion Mouse Model

Bu protokol, in vivo osteoklast aktivitesini kontrol eden kritik genleri anlamak için kurallı bir yöntemi açıklar. Bu yöntem, iskelet fenotipini analiz etmek için transgenik bir fare modeli ve bazı kurallı teknikler kullanır.

Koşullu Stat3 Silme Fare Modelinin İskelet Fenotip Analizi

Transgenic mouse models are powerful for understanding the critical genes controlling osteoclast differentiation and activity, and for studying mechanisms ...

skeletal-phenotype-analysis-conditional-stat3-deletion-mouse

Research

JoVE Journal

Biology

Skeletal Phenotype Analysis of a Conditional Stat3 Deletion Mouse Model

4.3K Görüntüleme.  National Clinical Research center of Stomatology. Bu protokol, in vivo osteoklast aktivitesini kontrol eden kritik genleri anlamak için kurallı bir yöntemi açıklar. Bu yöntem, iskelet fenotipini analiz etmek için transgenik bir fare modeli ve bazı kurallı teknikler kullanır.

Video: Skeletal Phenotype Analysis of a Conditional Stat3 Deletion Mouse Model

A Neuronal and Astrocyte Co-Culture Assay for High Content Analysis of Neurotoxicity

High Content Analysis (HCA) assays combine cells and detection reagents with automated imaging and powerful image analysis algorithms, allowing measurement of multiple cellular phenotypes within a single assay. In this study, we utilized HCA to develop a novel assay for neurotoxicity. Neurotoxicity assessment represents an important part of drug safety evaluation, as well as being a significant focus of environmental protection efforts. Additionally, neurotoxicity is also a well-accepted in vitro marker of the development of neurodegenerative diseases such as Alzheimer's and Parkinson's diseases. Recently, the application of HCA to neuronal screening has been reported. By labeling neuronal cells with &beta;III-tubulin, HCA assays can provide high-throughput, non-subjective, quantitative measurements of parameters such as neuronal number, neurite count and neurite length, all of which can indicate neurotoxic effects. However, the role of astrocytes remains unexplored in these models. Astrocytes have an integral role in the maintenance of central nervous system (CNS) homeostasis, and are associated with both neuroprotection and neurodegradation when they are activated in response to toxic substances or disease states. GFAP is an intermediate filament protein expressed predominantly in the astrocytes of the CNS. Astrocytic activation (gliosis) leads to the upregulation of GFAP, commonly accompanied by astrocyte proliferation and hypertrophy. This process of reactive gliosis has been proposed as an early marker of damage to the nervous system. The traditional method for GFAP quantitation is by immunoassay. This approach is limited by an inability to provide information on cellular localization, morphology and cell number. We determined that HCA could be used to overcome these limitations and to simultaneously measure multiple features associated with gliosis - changes in GFAP expression, astrocyte hypertrophy, and astrocyte proliferation - within a single assay. In co-culture studies, astrocytes have been shown to protect neurons against several types of toxic insult and to critically influence neuronal survival. Recent studies have suggested that the use of astrocytes in an in vitro neurotoxicity test system may prove more relevant to human CNS structure and function than neuronal cells alone. Accordingly, we have developed an HCA assay for co-culture of neurons and astrocytes, comprised of protocols and validated, target-specific detection reagents for profiling &beta;III-tubulin and glial fibrillary acidic protein (GFAP). This assay enables simultaneous analysis of neurotoxicity, neurite outgrowth, gliosis, neuronal and astrocytic morphology and neuronal and astrocytic development in a wide variety of cellular models, representing a novel, non-subjective, high-throughput assay for neurotoxicity assessment. The assay holds great potential for enhanced detection of neurotoxicity and improved productivity in neuroscience research and drug discovery.

This article describes a novel protocol and reagent set designed for sensitive measurement of neurotoxic effects of compounds and treatments on co-cultures of neurons and astrocytes using high content analysis. Results demonstrate that high content analysis represents an exciting novel technology for neurotoxicity assessment.

Nörotoksisite Yüksek İçerik Analizi Nöronal ve Astrosit Hücre Co-Kültür Testi

High Content Analysis (HCA) assays combine cells and detection reagents with automated imaging and powerful image analysis algorithms, allowing ...

Biyoloji

Analysis of the Development of a Morphological Phenotype as a Function of Protein Concentration in Budding Yeast

Gene deletion and protein overexpression are common methods for studying functions of proteins. In this article, we describe a protocol for analysis of phenotype development as a function of protein concentration at population and single-cell levels in Saccharomyces cerevisiae. Although this protocol is based on the overexpression of a protein, it can easily be adapted for morphological phenotypes dependent on suppression of protein expression. Our lab is interested in studying the signaling properties of the endocytic adaptor protein epsin. To that purpose we used a dominant negative approach in which we over-expressed the conserved Epsin N-Terminal Homology (ENTH) domain in order to interfere with the functions of endogenous epsin-2 (Ent2 or YLR206W). We observed that overexpression of the ENTH domain of Ent2 (ENTH2) in wild type cells led to a cell division defect that is dependent on the mislocalization of a family of scaffolding proteins, septins.

Gene deletion and protein overexpression are common methods for studying functions of proteins. In this article, we describe a protocol for analysis of phenotype development as a function of protein concentration at population and single-cell levels in Saccharomyces cerevisiae.

Geliştirici Maya Protein Konsantrasyonu bir işlev olarak Morfolojik Fenotip Kalkınma Analizi

Gene deletion and protein overexpression are common methods for studying functions of proteins. In this article, we describe a protocol for analysis of ...

In vivo Imaging and Therapeutic Treatments in an Orthotopic Mouse Model of Ovarian Cancer

Human cancer and response to therapy is better represented in orthotopic animal models. This paper describes the development of an orthotopic mouse model of ovarian cancer, treatment of cancer via oral delivery of drugs, and monitoring of tumor cell behavior in response to drug treatment in real time using in vivo imaging system. In this orthotopic model, ovarian tumor cells expressing luciferase are applied topically by injecting them directly into the mouse bursa where each ovary is enclosed. Upon injection of D-luciferin, a substrate of firefly luciferase, luciferase-expressing cells generate bioluminescence signals. This signal is detected by the in vivo imaging system and allows for a non-invasive means of monitoring tumor growth, distribution, and regression in individual animals. Drug administration via oral gavage allows for a maximum dosing volume of 10 mL/kg body weight to be delivered directly to the stomach and closely resembles delivery of drugs in clinical treatments. Therefore, techniques described here, development of an orthotopic mouse model of ovarian cancer, oral delivery of drugs, and in vivo imaging, are useful for better understanding of human ovarian cancer and treatment and will improve targeting this disease.

In vivo Imaging and Therapeutic Treatments in an Orthotopic Mouse Model of Ovarian Cancer

Orthotopic animal models of ovarian cancer replicate better human disease and therefore enhance our understanding of cancer progression and tumor response to therapy. A mouse model receives an intrabursal injection of luciferase-expressing ovarian tumor cells. Treatment is administered via oral gavage. Tumor growth is monitored by in vivo imaging system.

In vivo Görüntüleme ve Yumurtalık Kanseri Ortotopik Fare Modeli Tedavi Tedaviler

Human cancer and response to therapy is better represented in orthotopic animal models.  This paper describes the development of an orthotopic mouse model ...

Analysis of Skeletal Muscle Defects in Larval Zebrafish by Birefringence and Touch-evoke Escape Response Assays

Zebrafish (Danio rerio) have become a particularly effective tool for modeling human diseases affecting skeletal muscle, including muscular dystrophies1-3, congenital myopathies4,5, and disruptions in sarcomeric assembly6,7, due to high genomic and structural conservation with mammals8. Muscular disorganization and locomotive impairment can be quickly assessed in the zebrafish over the first few days post-fertilization. Two assays to help characterize skeletal muscle defects in zebrafish are birefringence (structural) and touch-evoked escape response (behavioral).
Birefringence is a physical property in which light is rotated as it passes through ordered matter, such as the pseudo-crystalline array of muscle sarcomeres9. It is a simple, noninvasive approach to assess muscle integrity in translucent zebrafish larvae early in development. Wild-type zebrafish with highly organized skeletal muscle appear very bright amidst a dark background when visualized between two polarized light filters, whereas muscle mutants have birefringence patterns specific to the primary muscular disorder they model. Zebrafish modeling muscular dystrophies, diseases characterized by myofiber degeneration followed by repeated rounds of regeneration, exhibit degenerative dark patches in skeletal muscle under polarized light. Nondystrophic myopathies are not associated with necrosis or regenerative changes, but result in disorganized myofibers and skeletal muscle weakness. Myopathic zebrafish typically show an overall reduction in birefringence, reflecting the disorganization of sarcomeres.
The touch-evoked escape assay involves observing an embryo&#39;s swimming behavior in response to tactile stimulation10-12. In comparison to wild-type larvae, mutant larvae frequently display a weak escape contraction, followed by slow swimming or other type of impaired motion that fails to propel the larvae more than a short distance12. The advantage of these assays is that disease progression in the same fish type can be monitored in vivo for several days, and that large numbers of fish can be analyzed in a short time relative to higher vertebrates.

The zebrafish is now an established and powerful tool for modeling muscular dystrophies, congenital myopathies, and related neuromuscular diseases. Birefringence and touch-evoked escape behavior are two common noninvasive assays used to determine the degree of muscular disorganization and locomotive impairment of zebrafish embryos during early development.

Zebrafish (Danio rerio) have become a particularly effective tool for modeling human diseases affecting skeletal muscle, including muscular ...

Alternative Cultures for Human Pluripotent Stem Cell Production, Maintenance, and Genetic Analysis

Human pluripotent stem cells (hPSCs) hold great promise for regenerative medicine and biopharmaceutical applications. Currently, optimal culture and efficient expansion of large amounts of clinical-grade hPSCs are critical issues in hPSC-based therapies. Conventionally, hPSCs are propagated as colonies on both feeder and feeder-free culture systems. However, these methods have several major limitations, including low cell yields and generation of heterogeneously differentiated cells. To improve current hPSC culture methods, we have recently developed a new method, which is based on non-colony type monolayer (NCM) culture of dissociated single cells. Here, we present detailed NCM protocols based on the Rho-associated kinase (ROCK) inhibitor Y-27632. We also provide new information regarding NCM culture with different small molecules such as Y-39983 (ROCK I inhibitor), phenylbenzodioxane (ROCK II inhibitor), and thiazovivin (a novel ROCK inhibitor). We further extend our basic protocol to cultivate hPSCs on defined extracellular proteins such as the laminin isoform 521 (LN-521) without the use of ROCK inhibitors. Moreover, based on NCM, we have demonstrated efficient transfection or transduction of plasmid DNAs, lentiviral particles, and oligonucleotide-based microRNAs into hPSCs in order to genetically modify these cells for molecular analyses and drug discovery. The NCM-based methods overcome the major shortcomings of colony-type culture, and thus may be suitable for producing large amounts of homogeneous hPSCs for future clinical therapies, stem cell research, and drug discovery.

Here, we present human pluripotent stem cell (hPSC) culture protocols, based on non-colony type monolayer (NCM) growth of dissociated single cells. This new method, utilizing Rho-associated kinase inhibitors or the laminin isoform 521 (LN-521), is suitable for producing large amounts of homogeneous hPSCs, genetic manipulation, and drug discovery.

İnsan Pluripotent için alternatif Kültürleri Hücre Üretim, Bakım ve Genetik Analizi Kök

Human pluripotent stem cells (hPSCs) hold great promise for regenerative medicine and biopharmaceutical applications. Currently, optimal culture and ...

Detection and Analysis of DNA Damage in Mouse Skeletal Muscle In Situ Using the TUNEL Method

Terminal deoxynucleotidyl transferase (TdT) deoxyuridine triphosphate (dUTP) nick end labeling (TUNEL) is the method of using the TdT enzyme to covalently attach a tagged form of dUTP to 3&#8217; ends of double- and single-stranded DNA breaks in cells. It is a reliable and useful method to detect DNA damage and cell death in situ. This video describes dissection, tissue processing, sectioning, and fluorescence-based TUNEL labeling of mouse skeletal muscle. It also describes a method of semi-automated TUNEL signal quantitation. Inherent normal tissue features and tissue processing conditions affect the ability of the TdT enzyme to efficiently label DNA. Tissue processing may also add undesirable autofluorescence that will interfere with TUNEL signal detection. Therefore, it is important to empirically determine tissue processing and TUNEL labeling methods that will yield the optimal signal-to-noise ratio for subsequent quantitation. The fluorescence-based assay described here provides a way to exclude autofluorescent signal by digital channel subtraction. The TUNEL assay, used with appropriate tissue processing techniques and controls, is a relatively fast, reproducible, quantitative method for detecting apoptosis in tissue. It can be used to confirm DNA damage and apoptosis as pathological mechanisms, to identify affected cell types, and to assess the efficacy of therapeutic treatments in vivo.

Detection and Analysis of DNA Damage in Mouse Skeletal Muscle In Situ Using the TUNEL Method

This video describes dissection, tissue processing, sectioning, and fluorescence-based TUNEL labeling of mouse skeletal muscle. It also describes a method for semi-automated analysis of TUNEL labeling.

Fare İskelet Kası Algılama ve DNA Hasar Analizi<em&gt; In Situ</em&gt; TÜNEL Yöntemiyle

Terminal deoxynucleotidyl transferase (TdT) deoxyuridine triphosphate (dUTP) nick end labeling (TUNEL) is the method of using the TdT enzyme to covalently ...

Cryosectioning of Contiguous Regions of a Single Mouse Skeletal Muscle for Gene Expression and Histological Analyses

With this method, consecutive cryosections are collected to enable both microscopy applications for tissue histology and enrichment of RNA for gene expression using adjacent regions from a single mouse skeletal muscle. Typically, it is challenging to achieve adequate homogenization of small skeletal muscle samples because buffer volumes may be too low for efficient grinding applications, yet without sufficient mechanical disruption, the dense tissue architecture of muscle limits penetration of buffer reagents, ultimately causing low RNA yield. By following the protocol reported here, 30 &#956;m sections are collected and pooled allowing cryosectioning and subsequent needle homogenization to mechanically disrupt the muscle, increasing the surface area exposed for buffer penetration. The primary limitations of the technique are that it requires a cryostat, and it is relatively low throughput. However, high-quality RNA can be obtained from small samples of pooled muscle cryosections, making this method accessible for many different skeletal muscles and other tissues. Furthermore, this technique enables matched analyses (e.g., tissue histopathology and gene expression) from adjacent regions of a single skeletal muscle so that measurements can be directly compared across applications to reduce experimental uncertainty and to reduce replicative animal experiments necessary to source a small tissue for multiple applications.

Consecutive cryo-sections are collected to enable histological applications and enrichment of RNA for gene expression measurements using adjacent regions from a single mouse skeletal muscle. High-quality RNA is obtained from 20 - 30 mg of pooled cryosections and measurements are directly compared across applications.

Gen İfade ve histolojik Analizler Tek Fare İskelet Kası bitişik Bölgeler Cryosectioning

With this method, consecutive cryosections are collected to enable both microscopy applications for tissue histology and enrichment of RNA for gene ...

Trans-inner Cell Mass Injection of Embryonic Stem Cells Leads to Higher Chimerism Rates

In an effort to increase efficiency in the creation of genetically modified mice via ES Cell methodologies, we present an adaptation to the current blastocyst injection protocol. Here we report that a simple rotation of the embryo, and injection through Trans-Inner cell mass (TICM) increased the percentage of chimeric mice from 31% to 50%, with no additional equipment or further specialized training. 26 different inbred clones, and 35 total clones were injected over a period of 9 months. There was no significant difference in either pregnancy rate or recovery rate of embryos between traditional injection techniques and TICM. Therefore, without any major alteration in the injection process and a simple positioning of the blastocyst and injecting through the ICM, releasing the ES cells into the blastocoel cavity can potentially improve the quantity of chimeric production and subsequent germline transmission.

Here we present a protocol to increase chimera production without the use of new equipment. A simple orientation change of the embryo for injection can increase the number of embryos produced, and potentially reduce the timeline to germline transmission.

Trans-iç hücre embriyonik kök hücrelerin kitle enjeksiyon daha yüksek Chimerism oranları için yol açar.

In an effort to increase efficiency in the creation of genetically modified mice via ES Cell methodologies, we present an adaptation to the current ...

Mass Spectrometry Analysis to Identify Ubiquitylation of EYFP-tagged CENP-A (EYFP-CENP-A)

Studying the structure and the dynamics of kinetochores and centromeres is important in understanding chromosomal instability (CIN) and cancer progression. How the chromosomal location and function of a centromere (i.e., centromere identity) are determined and participate in accurate chromosome segregation is a fundamental question. CENP-A is proposed to be the non-DNA indicator (epigenetic mark) of centromere identity, and CENP-A ubiquitylation is required for CENP-A deposition at the centromere, inherited through dimerization between cell division, and indispensable to cell viability.
Here we describe mass spectrometry analysis to identify ubiquitylation of EYFP-CENP-A K124R mutant suggesting that ubiquitylation at a different lysine is induced because of the EYFP tagging in the CENP-A K124R mutant protein. Lysine 306 (K306) ubiquitylation in EYFP-CENP-A K124R was successfully identified, which corresponds to lysine 56 (K56) in CENP-A through mass spectrometry analysis. A caveat is discussed in the use of GFP/EYFP or the tagging of high molecular weight protein as a tool to analyze the function of a protein. Current technical limit is also discussed for the detection of ubiquitylated bands, identification of site-specific ubiquitylation(s), and visualization of ubiquitylation in living cells or a specific single cell during the whole cell cycle.
The method of mass spectrometry analysis presented here can be applied to human CENP-A protein with different tags and other centromere-kinetochore proteins. These combinatory methods consisting of several assays/analyses could be recommended for researchers who are interested in identifying functional roles of ubiquitylation.

Mass Spectrometry Analysis to Identify Ubiquitylation of EYFP-tagged CENP-A EYFP-CENP-A

CENP-A ubiquitylation is an important requirement for CENP-A deposition at the centromere, inherited through dimerization between cell division, and indispensable to cell viability. Here we describe mass spectrometry analysis to identify ubiquitylation of EYFP-tagged CENP-A (EYFP-CENP-A) protein.

EYFP etiketli CENP-A (EYFP-CENP-A) Ubiquitylation tanımlamak için Kütle Spektrometresi Analizi

Studying the structure and the dynamics of kinetochores and centromeres is important in understanding chromosomal instability (CIN) and cancer ...

A Mouse Model of Lumbar Spine Instability

Intervertebral disc degeneration (IDD) is a common pathological change leading to low back pain. Appropriate animal models are desired for understanding the pathological processes and evaluating new drugs. Here, we introduced a surgically induced lumbar spine instability (LSI) mouse model that develops IDD starting from 1 week post operation. In detail, the mouse under anesthesia was operated by low back skin incision, L3&#8211;L5 spinous processes exposure, detachment of paraspinous muscles, resection of processes and ligaments, and skin closure. L4&#8211;L5 IVDs were chosen for the observation. The LSI model develops lumbar IDD by porosity and hypertrophy in endplates at an early stage, decrease in intervertebral disc volume, shrinkage in nucleus pulposus at an intermediate stage, and bone loss in lumbar vertebrae (L5) at a later stage. The LSI mouse model has the advantages of strong operability, no requirement of special equipment, reproducibility, inexpensive, and relatively short period of IDD development. However, LSI operation is still a trauma that causes inflammation within the first week post operation. Thus, this animal model is suitable for study of lumbar IDD.

We developed a lumbar intervertebral disc degeneration mouse model by resection of L3&#8211;L5 spinous processes along with supra- and inter-spinous ligaments and detachment of paraspinous muscles.

Bel Omurgası Kararsızlığının Fare Modeli

Intervertebral disc degeneration (IDD) is a common pathological change leading to low back pain. Appropriate animal models are desired for understanding ...