Yazarlar laboratuvar İş USDA-/ CSREES / NRICGP (2007-35203-18213) tarafından desteklenen; NIH / NCRR (2 R01 RR17359-06), NIH / NIEHS (1 R21 ES014856-01A2) ve Alberta yenilikler yaratır - Sağlık Çözümleri.

BÖLÜM farelerde A. Germ hücre nakli 1. , Alıcının fareler of Hazırlanışı <ol><li> Alıcılar donör testis hücreleri (donörlerden ya da bağışıklık eksikliği ya da genetik olarak uyumlu) bağışıksal hoşgörülü olmalıdır. </li><li> Alıcılar ya doğal olarak spermatogenez yoksun (örneğin W / W v fareler) veya endojen germ hücrelerinin tüketilmiş olması gerekir. Germ hücre tükenmesi,, ışınlama ya da bu tür Myleran Film Tablet&#39;ın gibi kemoterapötik uyuşturucular kullanıcısı tarafından başarılabilir uygulanmaz edebilirsiniz. , Bu protokol In, biz,,, Myleran Film Tablet&#39;ın ile birlikte alıcının fareler hazırlarken of yöntemini açıklamak. </li><li> 4-6 hafta boyunca yaş ip busulfandan enjeksiyonu alıcılara davranın. Busulfandan optimal doz suşu bağlıdır. , Olarak, yaygın olarak olarak kullanılan alıcının suşlar In, 40-50 mg / kg &#39;dan&#39; of, bir doz,,,,, endojen germ hücreleri (örn.: 44, nude fareler için mg / kg &#39;, 50 mg /, B6/129 F1 alıcıların için kg&#39;). Deplete etmek yeterli bir konumundadır </li><li> DMSO Busulfan Tozun erimesi ve daha sonra steril distile su t eşit hacimde ekleyinyapmak Kullanıcısına, 4 mg / ml &#39;&#39; of, bir nihai bir bir konsantrasyon kaçınılır. 35-40 ° C daha önce yağış busulfandan önlemek için kullanabileceğiniz çözüm sıcak tutun. Yağış görülür çözüm atın. </li><li> Olarak, Myleran Film Tablet&#39;ın tedavi sonra,,, en azından at birini ayın alıcıların kullanmadan önce, izin vermek. Recipients,, 1 ayın, ve 3 ay önce yazılan mesaja-Myleran Film Tablet&#39;ın tedavi. Tarihleri ​​arasında kullanılan uygulanmaz edebilirsiniz. </li></ol> 2. Mikroenjeksiyon pipetler hazırlanması <ol><li> 1,0 mm dış çapı, 0,75 mm iç çap ve 3 inç uzunluğu ile borosilikat cam pipetler (kılcal tüpler) seçin. </li><li> Cam pipetler Sigmacote Siliconize, metanol ile pipetler çalkalayın ve kuru bir darbe. </li><li> Pipetler bir pipetle Fuari&#39;ndaki kullanarak çekin. , Farklı pipettir çektirmenin makinelerde,, farklı ayarlarını, bu nedenle, bir birkaç ayarlarını,, sınamak, var. Genellikle olarak,,,,, enükleasyon operasyonu uygulandı pipetlerimiz yaptıktan için kullanılan olduğunu etmek &#39;e benzer, bir ayarı, çalışmak olabilir. </li><li> ,,, Yaklaşık olarak 50 mikron of, bir çapı başarmak etmek kullanmak etmek, öncesinde,, dissekan mikroskop altında pipettir bir ip uçları Breakucunda. </li></ol> 3. Nakli için donör hücrelerinin hazırlanması <ol><li> Donör suşu Choise çalışılan deneysel soru üzerine bağlıdır. Spermatogenez ölçümü son nokta olarak kullanılması durumunda, Lac-Z gibi kolayca tanımlanabilen genetik işaretleyici ile donörlerin (örneğin B6.129S7 Gt (ROSA) Jackson Laboratory 26Sor / J) veya GFP (örneğin C57BL/6-Tg ( Jackson Laboratory CAG-EGFP in) 1Osb / J). </li><li> 7 mg / ml &#39;&#39; at,, DMEM in, DNase of, mg / ml &#39;, Şu (4), tripsin-EDTA ile of ml&#39; (% 0.25 &#39;lik, tripsin plus 1mM valdecoxib ile yapılan EDTA ile), ve 2 adet ml&#39; 1 - at, (7), DMEM in collagenase of ml &#39;hazırlayın. Bu miktar 2 donör fare testis sindirerek içindir. </li><li> Aseptik testis toplayın ve tunika albuginea kaldırmak. Yayılma seminifer enzimatik sindirimi kolaylaştırmak için ince forseps ile yavaşça tübüller. </li><li> 5-10 dakika, ve sık sık ajitasyon için 37 ° C&#39;de inkübe kollajenaz içine transfer tübüller. </li><li> Temizlik tübüllerin, iplik eğirme (3 min için: 200-300 g) ve, #, PBS w / o Ca 2 bölgesindeki re-süspansiyona alma kullanıcısı tarafından iki kat daha yıkayın.. + + </s&gt; kadar. </li><li> Tripsin-EDTA tübüller yeniden askıya alma ve yapışkan ve bulanık hale gelene kadar salla. Tübüllerin 1-2 dakika içinde meydana gelen 37 ° C gibi sindirim izleyin. </li><li> DNaz ekleyin ve iyice çalkalanır. 1-2 dakika boyunca inkübe edin. </li><li> ,,, Enzimler of eylem durdurmak etmek 3 ml &#39;of FBS ekle. </li><li> Hücre / doku parçalarını kaldırmak için, 40-70 mikron gözenek büyüklüğüne sahip bir naylon mesh kullanarak Filtre hücre süspansiyonu. </li><li> ,, 5 min Şu aramanız için 600 g için at hücreleri Collect. Ve bu konuda kurulacak güçlü,, DMEM (&lt;100 ul&#39;lik) of, bir küçük bir bir miktarını in re-askıya alma hücreleri. </li><li> Hücreleri saymak ve istenen nihai konsantrasyonu (genellikle 100 x 10 6) ses seviyesini ayarlamak. Kullanmadan önce buz üzerinde hücre süspansiyonu tutun. </li><li> Her, busulfan-tedavi edilen fare testisi hücre süspansiyonu sadece 10-15 ul dolacaktır. Potansiyel atık ve sızıntı nedeniyle, testis başına 30-50 ul hedefliyoruz. </li></ol> 4. Nakli işlem (Şekil 1) <ol><li> Onaylı protokolleri takip alıcıları uyuşturan. </li><li> Dorsal içinde Yeripozisyonda ve cerrahi karın bölgesi hazırlamak. Karın duvarı açığa çıkarmak için bir ~ 1cm orta hat karın kesisi olun. Yanlışlıkla karın içi organların zarar vermekten kaçınmak ve periton boşluğuna maruz karın duvarı orta hattan ~ 0.5 cm kesi yapmaya devam beyaz çizgi noktada küçük forseps kullanarak karın duvarı kaldırın. </li><li> Karın duvarı tutmak için bir iris forseps kullanın ve yağ pedleri, periton boşluğuna epididim ve testis bağlı aramak için iris forsepsinin başka bir çifti kullanmak. Testis yavaşça dışarıya alınır kadar yağ yastığı, testiküler arter ve epididim çekin ve net bir şekilde görülebiliyor. Bir anda bir testis üzerine çalışın. </li><li> Daha iyi bir görsel kimlik (isteğe bağlı) yağ yastığı / testis altında otoklavlanmış indeks kartları ile yapılan ince bir steril örtüyü yerleştirin. Drape Ayrıca sıvı absorbe etmek için çalışır. </li><li> ,,,, Hücre süspansiyon içine internetin Tripan Blue dye ile of, bir drop ekle ve dikkatli bir şekilde,, Polietilen içine hücre süspansiyon da yüklenemedi.lene hortumunu,, bir 1 ml &#39;şırınga etmek bağlandığında. Tüp içine çekti pipet pipet içine hücre süspansiyonu enjektöre basınç uygulayarak takın ve hafifçe zorlamak. </li><li> Efferent kanalları (testisin epididim bağlayan) belirlenmesi ve yavaşça kanalları çevresinde yağ doku kaldırmak. Çevrelerindeki kanalları ve membran saydam dikkatli bir şekilde çalışın. </li><li> Efferent kanalların paket içinde dikkatlice bir kanal içine pipet eklemek, yavaşça, testis doğru birkaç mm geçiriniz. </li><li> , Enjeksiyon pipet hareket kaçınarak ulaşmak için şırınga, yavaşça doldurmaya başlar askıya rete testis ve seminifer tübül içine akar sağlamak için şırınga pistonu bastırın. </li><li> Hücre süspansiyonu enjeksiyon hızı ve akış parmak basıncı ile düzenlenir. Basıncındaki ani artış kaçının; tübüllerinde süspansiyon hareketini izlemek. </li><li> Testis bec başlamadan önce hemen hemen tüm yüzey tübüller doldurulmuş enjeksiyon Durduriskemik Ome. </li><li> Testis karın boşluğuna getirin. Kontralateral testis üzerine işlemi tekrarlayın. 6-0 ipek sütür ile karın duvarı dikerek ve deri metal yara klipler ile kapatın. Tam iyileşme kadar bir ısınma pad üzerinde fareler izleyin. </li></ol> 5. Alıcı testislerin analizi <ol><li> Analizden önce transplantasyon sonrası 2-4 ay izin verin. </li><li> Hayvan bakımı ve kullanım yönergelerine göre, alıcı fare Euthanize, PBS içine testis ve epididim toplamak. , It,, endojen bir beta-galactosidase aktivitesi sahiptir gibi,, bir Lac-Z arasından transgenic donör strain, kullanılan&gt; edilmiştir&gt; gelmiştir Ne zaman, epididimis,,, bir pozitif bir bir boyanma kontrolü gibi kullanılmalıdır edebilirsiniz. </li><li> Lac-Z boyama ile donör hücrelerinin varlığı tespiti için 1-2 saat, 4 testis düzeltmek ° C PBS içinde% 4 paraformaldehit (PFA). ,, LacZ şehrindeki In-room sıcaklık at 3 x 30, min durulayın buffer durulama. </li><li> ,, LacZ boyanma bir çözüm in, (37) ° C at bir gecede inkübe edin. Testis,,, bir, bütünün gibi ya da dispersleme sonra, boyand uygulanmaz edebilirsiniztübüller. </li><li> Saptamak ve% 10 nötr tamponlu formalin depolamak. Bölümleri gerekiyorsa karşı leke olarak nötr hızlı kırmızı kullanın. </li></ol> KISIM B. Ektopik farelerde xenografting (Dobrinski ve rathi 15, 2008 da, ve Rodriguez-Sosa ve ark. Ark, Yorumdan ... 2011 16 kişi). 1. Donör doku toplanması <ol><li> Hadım testis dokusu elde etmek ya da bir bağış erkek biyopsi yapıldı. </li><li> PBS veya biyopsi kültür ortamında steril şartlarda muhafaza Yer testis. </li><li> Buz ve laboratuvar taşıma toplanan doku tutun. </li></ol> 2. , Donör doku of Hazırlanışı <ol><li> Sterilite korumak için doku kültürü kaputu gerçekleştirin. </li><li> 2-3 kez PBS ile bir kültür kabına aktarmadan önce buz gibi soğuk PBS içeren antibiyotikler her testisteki yıkayın. Biyopsi durumda, buz kültürü medi testis parçaları ile 2-3 kez yıkamakum,, 2 min at 150 g for iplik eğirme ve,, taze - buz-soğuk algınlığı bir kültürü medium in resuspending kullanıcısı tarafından antibiyotikler ihtiva eden ve eğitimi. </li><li> Sağlam testis için, yüzeyi boyunca bir kesi yaparak tunika vaginalis testis kaldırmak ve a&#39;ya. Testis tüm ek yapılar (spermatik kordon, epididim, bağ dokusu) çıkar. PBS ile bir kültür kabına soğuk PBS ve transfer testisler bir kez yıkayın. </li><li> Testisin tunika albuginea, bir neşter bıçak ve bir makas kullanarak dikkatlice çıkarın. Testis çok küçükse, tunika diğer ucunda küçük forseps bir çift ile tunika tutarken, bir ucunda yapılan küçük bir kesi yoluyla tunika testis dokusu sıkma kaldırılabilir. </li><li> , Testisin büyüklüğüne bağlı olarak ya da tüm testis dokusu yaklaşık 1 küçük parçalar halinde kesilir. Kavisli bir forseps ve bir neşter bıçağı, ya da testis dokusu büyük parçalar kullanarak boyutu 2 mm 3 ilk testisten kaldırılır ve olabilir küçük içine kesinparçalar. All, bu,,, ice-soğuk algınlığı bir kültürü medium in ve,,, bir küçük bir bir kültürü çanağı (60 X15 mm), in steril bir koşullar altında yapılabilir uygulanmaz gerektiğini. </li><li> Aşılama kadar buz üzerinde küçük kültür-yemekleri buz gibi kültür ortamına hazırlanan doku parçaları aktarın. </li></ol> 3. Kastrasyon ve alıcı fare <ol><li> Yukarıdaki gibi fare uyuşturan ve% 70 Etanol ve Betadine solüsyonu ile silinerek, saç (çıplak farelerin gerekli değildir) takarak, steril cerrahi saha hazırlamak. </li><li> Karın duvarı açığa çıkarmak için 0.5 -1 cm ventral orta hat cilt insizyonu olun. </li><li> Dikkatle, Parta, 4,2-4,3 açıklanan olarak testis, testis arter ve epididim maruz kalmaktadır. </li><li> Künt diseksiyon ile, gubernakulum Epididimisin kuyruğu ayırın. </li><li> Testiküler arter Ligate ve ipek ile kan damarları ile birlikte vas deferens ve testis ve ligatür arasındaki kesim tarafından bağlanan yapılar bölümü. </li><li> Seco için aynı işlemi tekrarlayın.nd testis. </li><li> Karın duvarı, bir ya da iki cerrahi dikiş ile dikin. </li><li> Bir ya da iki Michel klipler ile cilt kesisi kapatın. </li></ol> 4. Xenografting Ektopik <ol><li> Fare ventral pozisyonda yerleştirin ve yukarıdaki gibi sırtında bir steril cerrahi alan hazırlamak. </li><li> Ekleneceği (genellikle 4-8/mouse) kaç greft bağlı olarak, geri fare orta çizginin her iki tarafında ~ 0.5 cm uzunluğunda cilt insizyonu olun. </li><li> Cilt kesisi bir sınır tutmak için forseps kullanın ve makas kullanırken bağ dokusu dışında alay subkutan boşluğu yapmak. </li><li> Başka bir iris forseps ile cilt kesisi sınır tutarak, bir iris forseps yerde derin subkutan boşluğuna testis dokusu bir parça kullanma. </li><li> Bir Michel klibi ile cilt kesisi kapatın ve anestezi kurtarmak başlayıncaya kadar ısıtma pedi fare tutmak. </li><li> Ek izolasyon ve işbirliği ile bir kafes fare aktarınver ve monitör fareler kadar tamamen iyileşti. </li></ol> 5. , Testis analizi ve sperm toplama xenogreftler Koleksiyonu <ol><li> Hayvan bakımı ve kullanım yönergelerine göre ana fare Euthanize ve kuyruk ve boyun açık cilt için çalışan arka cilt üzerinde orta hat cilt kesi yapmak. Bu,, subkutan doku on, ya da olarak şuradadır ya da,, cilt etmek iliştirilmiş edilebilir greftler açýklar. </li><li> Bir çift forseps ve bir makas kullanarak greft dikkatlice çıkarın. </li><li> Greft kurtarıldı, bireysel greftlerin büyüklüğü ve ağırlığı da rekor sayıda. </li><li> Farenin karın seminal veziküllerin Al ve aşılı doku testosteron üretiminin bir göstergesi olarak kendi ağırlığı kaydetmek. </li><li> Histolojik analiz için <ol class="lalpha"><li> Hacminde Bouin çözümü (ya da diğer fiksatif) içeren örnek şişesine xenogreftler askıya almasını xenograftlarının, 10x ~~ ve Flakon uygun etiket. </li><li> Üretmek% 70 etanol içinde tercihen 24 saat aralıklarla en az 3 kez yıkama ardından buzdolabında bir gece. </li><li> Işleme ve parafine gömülmesi için devam edin. </li></ol></li><li> Sperm toplama için <ol class="lalpha"><li> 300g az 1 dakika için onları aşağı eğirme ve kültür ortamı içeren antibiyotikler onları resuspending xenogreftler yıkayın. </li><li> Küçük parçalar halinde greftler 3 içeren bir doku kültürü çanak forseps ile dikkatlice kesin ve kıyma - 5 ml kültür ortamı. </li><li> 40 mikron hücre süzgecinden Filtre kıyılmış doku. </li></ol></li></ol> BÖLÜM C. Temsilcisi Sonuçlar 1. Germ hücreli Transplantation X-gal boyama gibi, alıcı testiste donör DGM&#39;lerden kolonizasyon lacZ transgen gibi genetik bir marker taşıyan spermatogonial kök hücreleri nakledilen donör hücre süspansiyonu varsa sem ayırt edici mavi bölümleri ile görülebilmesiiniferous 2-3 ay sonra transplantasyon sonrası 3 (Şekil 2A) tübüller. , Eşleştirilmiş tek veya küçük gruplar mavi hücreleri bir ağ, iyi kurulmuş bir koloni biter hem de iki veya daha fazla, daha merkeze yakın mavi hücre katmanları ve lekeli bölgelerde göreceli olarak daha zayıf kesimlerinin tamamen dolu uzun bir koyu mavi streç olmalıdır. , 3 (Şekil 2B) görünürdeki konumundadır. Koyu mavi seminifer tübül bölgenin köklü bir kesit ortaya çıkarmak ve çeşitli farklılaşma aşamaları (Şekil 2C) mavi germ hücreleri ile iyi organize spermatogenez. 2. Xenografting testis dokusu Transplantasyonu sonrası testis dokusu canlılığını donör gelişimsel aşaması ile ters orantılıdır; yetişkin doku 17-21 dejenere ve ölmek için yüksek bir eğilim gösterirken, en iyi sonucu, yeni doğan erkek doku kullanıldığı zaman elde edilir. Genellikle, xenograft başarısı azalır donör doku bana uğrar gibispermatogenez ilk dalga iosis. Olgunlaşmamış donör doku ve tam spermatogenez tam gelişmesini Zaman türe özgü olup, in situ testis ile karşılaştırıldığında genellikle biraz daha kısa. Seminifer tübüller tam spermatogenez sayısı türlere göre değişkendir. Koyun iken, keçi ve domuz sayısı sığır ve kedilerde% 50 daha büyük olduğunu,% 15 den az 14 (Şekil 3). <img alt="Şekil 1," src="/files/ftp_upload/3545/3545fig1.jpg" /> Şekil 1. Germ hücre nakli prosedürü Yer anestezi sonrası dorsal pozisyonda alıcılar ve bir ~ 0.4-inç orta hat karın kesisi (A) yapmak. Testis, epididim ve testis bağlı yağ yastığı çekildiğini ortaya çıkarmak ve daha iyi bir görsel kimlik (B) yağ yastığı / testis altında ince bir steril örtüyü yerleştirin. Testis ve epididim, yağ yastığı gömülü efferent kanalları (C, D, E) görülebilir böylece yerleştirin. Kimlikvarlık efferent kanalları ve yavaşça kanalları çevresinde yağ doku kaldırmak. Renkli kağıt veya plastik bir parça daha iyi görselleştirme (G) kanalları altına yerleştirilebilir. ,,,, Içine the pipettir (H) kanallarında ve load cell süspansiyon of boyutu etmek göre,,, pipettir bahşiş Break ya da öğütmek. Efferent kanalların paket içinde dikkatlice bir kanal içine pipet eklemek testis (H ok pipet enjeksiyon ve iş parçacığı yönünü gösterir) doğru, yavaşça birkaç mm geçiriniz. Seminifer tübüller içine başarılı bir enjeksiyon ile testis (I) gösterilir. Ts: Testis; Ep: epididimden. <img alt="Şekil 2," src="/files/ftp_upload/3545/3545fig2.jpg" /> Şekil 2. Germ hücre transplantasyonu A) X-gal ile boyanmış bir enjekte testis Temsilcisi sonuç bulundu. ,, Seminifer tubüllerin mavi segmentleri, transgenik donör DGM&#39;lerden (lacZ transgeni) kurulan koloniler temsil eder. 3 ay transplantasyon sonrası bir SSC koloni B) Yüksek büyütme. The merkezi koyu mavi bölge ucunda tam spermatogenez ve soluk mavi bölgeleri temsil büyüyen uzantısı koloninin temsil eder. C), koyu mavi seminifer tübül histolojik bölüm (3 ay transplantasyon sonrası) iyi organize spermatogenez gösterir. B ve C Ölçek bar 200 mikron ve 30 mikron. (Şekil, Annu Rev Hücre Dev Biol adapte 2008;. 24:263-86 ve Biol REPROD 1999 Haz. 60 (6) :1429-36). <img alt="Şekil 3," src="/files/ftp_upload/3545/3545fig3.jpg" /> Şekil 3,. Ektopik bağışık yetmezliği olan farelere içine büyük hayvanlardan elde edilen olgunlaşmamış testis dokusu xenografting, bağışık yetmezliği olan farelere dorsal cilt altında (A) nakledilen olgunlaşmamış donör testis Fragments (~ 1 mm 3) hayatta ve fare Gonadotropinlerle cevap mümkün. Bir sonucu As, testis dokusundaki değişikliklerin,,,,,, fertilizasyon, yetkili sperm (Yatak-), of formasyonu de dahil olmak üzere, komple kalkınma undergoes. , Testis xenogreftler topladıktan sonra (C)onlar,,,, analizi için ya da,,,, ICSI (E) ve embriyo üretim (F) için sperm (-Ge) edinme etmek. kullanılan uygulanmaz edebilirsiniz. Barlar eşit 50 mikron (B, E ve F) veya 10 mikron (D). (Modifiye Rodriguez-Sosa ve Dobrinksi 14, 2009).

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M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sertoli+cells+dictate+spermatogonial+stem+cell+niches+in+the+mouse+testis.">Sertoli cells dictate spermatogonial stem cell niches in the mouse testis.</a> Biology of Reproduction. 84 (4), 639-639 (2011).</li><li>Nagano, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Homing+efficiency+and+proliferation+kinetics+of+male+germ+line+stem+cells+following+transplantation+in+mice.">Homing efficiency and proliferation kinetics of male germ line stem cells following transplantation in mice.</a> Biology of Reproduction. 69 (2), 701-701 (2003).</li><li>Kubota, H., Avarbock, M. R., Brinster, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Growth+factors+essential+for+self-renewal+and+expansion+of+mouse+spermatogonial+stem+cells.">Growth factors essential for self-renewal and expansion of mouse spermatogonial stem cells.</a> Proceedings of the National Academy of Sciences of the United States of America. 101 (47), 16489-16489 (2004).</li><li>Oatley, J. M., Avarbock, M. R., Telaranta, A. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identifying+genes+important+for+spermatogonial+stem+cell+self-renewal+and+survival.">Identifying genes important for spermatogonial stem cell self-renewal and survival.</a> Proceedings of the National Academy of Sciences of the United States of America. 103 (25), 9524-9524 (2006).</li><li>Dobrinski, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Germ+cell+transplantation+and+testis+tissue+xenografting+in+domestic+animals.">Germ cell transplantation and testis tissue xenografting in domestic animals.</a> Animal Reproduction Science. 89 (1-4), 137-137 (2005).</li><li>Dobrinski, I., Avarbock, M. R., Brinster, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transplantation+of+germ+cells+from+rabbits+and+dogs+into+mouse+testes.">Transplantation of germ cells from rabbits and dogs into mouse testes.</a> Biology of Reproduction. 61 (5), 1331-1331 (1999).</li><li>Honaramooz, A., Snedaker, A., Boiani, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sperm+from+neonatal+mammalian+testes+grafted+in+mice.">Sperm from neonatal mammalian testes grafted in mice.</a> Nature. 418 (6899), 778-778 (2002).</li><li>Rodriguez-Sosa, J. R., Dobrinski, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recent+developments+in+testis+tissue+xenografting.">Recent developments in testis tissue xenografting.</a> Reproduction. 138 (2), 187-187 (2009).</li><li>Dobrinski, I., Rathi, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ectopic+grafting+of+mammalian+testis+tissue+into+mouse+hosts.">Ectopic grafting of mammalian testis tissue into mouse hosts.</a> Methods in Molecular Biology. 139, 450-450 (2008).</li><li>Rodriguez-Sosa, J. R., Schlatt, S., Dobrinski, I., Seli, E., Agarwal, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Testicular+tissue+transplantation+for+fertility+preservation.">Testicular tissue transplantation for fertility preservation.</a> Fertility Preservation: Emerging Technologies and Clinical Applications. , 331-331 (2011).</li><li>Rathi, R., Honaramooz, A., Zeng, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Germ+cell+fate+and+seminiferous+tubule+development+in+bovine+testis+xenografts.">Germ cell fate and seminiferous tubule development in bovine testis xenografts.</a> Reproduction. 130 (6), 923-923 (2005).</li><li>Rathi, R., Honaramooz, A., Zeng, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Germ+cell+development+in+equine+testis+tissue+xenografted+into+mice.">Germ cell development in equine testis tissue xenografted into mice.</a> Reproduction. 131 (6), 1091-1091 (2006).</li><li>Kim, Y., Selvaraj, V., Pukazhenthi, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+donor+age+on+success+of+spermatogenesis+in+feline+testis+xenografts.">Effect of donor age on success of spermatogenesis in feline testis xenografts.</a> Reproduction, Fertility, and Development. 19 (7), 869-869 (2007).</li><li>Arregui, L., Rathi, R., Zeng, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Xenografting+of+adult+mammalian+testis+tissue.">Xenografting of adult mammalian testis tissue.</a> Animal Reproduction Science. 106 (1-2), 65-65 (2008).</li><li>Schlatt, S., Honaramooz, A., Ehmcke, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Limited+survival+of+adult+human+testicular+tissue+as+ectopic+xenograft.">Limited survival of adult human testicular tissue as ectopic xenograft.</a> Human Reproduction. 21 (2), 384-384 (2006).</li><li>Shinohara, T., Avarbock, M. R., Brinster, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=beta1-+and+alpha6-integrin+are+surface+markers+on+mouse+spermatogonial+stem+cells.">beta1- and alpha6-integrin are surface markers on mouse spermatogonial stem cells.</a> Proceedings of the National Academy of Sciences of the United States of America. 96 (10), 5504-5504 (1999).</li><li>Kanatsu-Shinohara, M., Toyokuni, S., Shinohara, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CD9+is+a+surface+marker+on+mouse+and+rat+male+germline+stem+cells.">CD9 is a surface marker on mouse and rat male germline stem cells.</a> Biology of Reproduction. 70 (1), 70-70 (2004).</li><li>Ryu, B. Y., Orwig, K. E., Oatley, J. 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D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ETV5+is+required+for+continuous+spermatogenesis+in+adult+mice+and+may+mediate+blood+testes+barrier+function+and+testicular+immune+privilege.">ETV5 is required for continuous spermatogenesis in adult mice and may mediate blood testes barrier function and testicular immune privilege.</a> Annals of the New York Academy of Sciences. 1120, 144-144 (2007).</li><li>Zeng, W., Snedaker, A. K., Megee, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preservation+and+transplantation+of+porcine+testis+tissue.">Preservation and transplantation of porcine testis tissue.</a> Reproduction, Fertility and Development. 21 (3), 489-489 (2009).</li><li>Jahnukainen, K., Ehmcke, J., Hergenrother, S. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+cold+storage+and+cryopreservation+of+immature+non-human+primate+testicular+tissue+on+spermatogonial+stem+cell+potential+in+xenografts.">Effect of cold storage and cryopreservation of immature non-human primate testicular tissue on spermatogonial stem cell potential in xenografts.</a> Human Reproduction. 22 (4), 1060-1060 (2007).</li><li>Rathi, R., Zeng, W., Megee, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Maturation+of+testicular+tissue+from+infant+monkeys+after+xenografting+into+mice.">Maturation of testicular tissue from infant monkeys after xenografting into mice.</a> Endocrinology. 149 (10), 5288-5288 (2008).</li><li>Honaramooz, A., Li, M. W., Penedo, M. 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Çıkar çatışması ilan etti.

1. Germ hücreli Transplantation Germ hücre transplantasyonu (DGM) bir hücre popülasyonu spermatogonial kök hücrelerin varlığı kesin onay için sadece fonksiyonel ölçüm sağlar. Sadece DGM&#39;lerden ve ev bazal membran SSC niş kolonize ve donör kaynaklı spermatogenez başlatmak. Germ hücre transplantasyonu, DGM&#39;lerden benzeri görülmemiş bir şekilde çalışmak ve işlemek için mümkün kıldı. Teknik, DGM&#39;lerden 4, genetik manipülasyon yoluyla transgen...

<table border="1"><tbody><tr><td> Reaktif Adı </td><td> Şirket </td><td> Katalog numarası </td></tr><tr><td> Kollajenaz (tip IV) </td><td> Sigma </td><td> C5138 </td></tr><tr><td> Tripsin-EDTA </td><td> Invitrogen </td><td> 25200-056 </td></tr><tr><td> DNaseI </td><td> Sigma </td><td> DN25 </td></tr><tr><td> DMEM </td><td> Invitrogen </td><td> 31053-028 </td></tr><tr><td> Tripan mavisi KORUYUCU </td><td> Invitrogen </td><td> 15250-061 </td></tr><tr><td> Naylon örgü hücre süzgecinden </td><td> BD biyoloji </td><td> 352.340 (40μm) 352.350 (70μm) </td></tr><tr><td> Busulfan </td><td> Sigma </td><td> B2635 </td></tr><tr><td> İnce Wall Cam kılcal </td><td> Dünya Hassas Çalgı </td><td> TW 100-3 </td></tr><tr><td> BD intramedic plyethylene boru (PE100) </td><td> BD </td><td> CA-63018-725 </td></tr><tr><td> Ethicon 6-0 İpek Sütür </td><td> Ethicon </td><td> 706g </td></tr><tr><td> Yara klipler </td><td> BD </td><td> 427631 </td></tr><tr><td> Sigmacote </td><td> Sigma </td><td> SL2 </td></tr><tr><td> X-gal </td><td> Sigma </td><td> B4252 </td></tr><tr><td> Ferrocyanide Potasyum </td><td> Sigma </td><td> P9387 </td></tr><tr><td> Potasyum ferrisiyanürle </td><td> Sigma </td><td> P3667 </td></tr><tr><td> magnezyum klorür </td><td> Sigma </td><td> 208337 </td></tr><tr><td> sodyum deoksikolat </td><td> Sigma </td><td> D6750 </td></tr><tr><td> , N, N-Dimethylformamide </td><td> Sigma </td><td> D4551 </td></tr><tr><td> Igepal CA-630 </td><td> Sigma </td><td> 18896 </td></tr></tbody></table>

germ cell transplantation and testis tissue xenografting in mice

Germ hücre transplantasyonu 1994 1,2 Pennsylvania Üniversitesi&#39;nden Dr., Ralph Brinster ve arkadaşları tarafından geliştirilmiştir. Bu çığır açan çalışmalar alıcının hayvan 2 donör kaynaklı spermatogenez ve sperm üretimi, infertil alıcı fareler sonuçları seminifer tübüller içine bereketli donör farelerin germ hücreleri bu mikroenjeksiyon gösterdi. Kullanımı alıcı hayvanların 1 yavrular, donör haplotypein donör kaynaklı spermatogenez ve iletim bakteriyel β-galaktosidaz gen izin kimlik taşıyan donör erkek. Şaşırtıcı, seminifer tübül lümenine naklinden sonra, nakledilen germ hücreleri, spermatogonium 3 yer vardır bazal membran luminal bölmesini taşımak için başardık. Genellikle sadece DGM&#39;lerden alıcı testis ve spermatogenez yeniden kurmak niş kolonize olduğu kabul edilir. Bu nedenle, germ hücrelitransplantasyonu,, testisinde kök hücre niş okumak için ve olası spermatogonial kök hücre karakterize fonksiyonel bir yaklaşım sağlar. Bugüne kadar, germ hücre transplantasyonu, temel kök hücre biyolojisi aydınlatmak için önce germ hücrelerinin genetik manipülasyon yoluyla nakli 4,5 transgenik hayvanlar üretmek için, Sertoli cell-germ hücreli etkileşimi 6,7, SSC homing ve kolonizasyon 3 çalışmak için kullanılır. 8, yanı sıra gibi SSC kendi kendine-yenilenmesini değişiklik yapmış ve diferansiyasyon 9,10. Germ hücre nakli 11 iri türlere de mümkündür. Bu, ana uygulamalar doğurganlığın korunması, hayvan popülasyonlarının elit genetik yayılması ve bu teknik ile, spermatogenez ve SSC biyoloji çalışma lojistik, kemirgenlerde göre daha zor ve pahalı olduğu gibi, transgenik hayvan üretimi vardır. Farelerde colonizat sonuçları seminifer tübüller içine iri türlere germ hücrelerinin naklidonör hücrelerinin iyon ve spermatogonial genişleme değil, muhtemelen, filogenetik olarak uzak türler 12 germ hücreleri ile alıcı somatik hücre bölmesi uyumsuzluk nedeniyle tam farklılaşma. Alternatif bir yaklaşım çevreleyen somatik bölmesi ile birlikte büyük türlerinin germ hücre naklidir. Biz ilk dorsal deri altında bağışık yetmezliği olan farelere nakledilen olgunlaşmamış erkek testis dokusu küçük parçaları 13 döllenme yetkili sperm üretimi ile tam gelişmesi ancak hayatta geçmesi mümkün olduğunu, 2002 yılında bildirilmiştir. O zamandan beri birçok türün başarılı olmak için xenografting testis dokusunda gösterilmiştir ve farelerin 14 büyük hayvanların testis gelişimi ve spermatogenez incelemek için değerli bir alternatif olarak ortaya çıkmıştır.

Watch this Scientific Journal Video about Germ Cell Transplantation and Testis Tissue Xenografting in Mice at JoVE.com

Germ Cell Transplantation and Testis Tissue Xenografting in Mice

Germ hücre nakli ve testis dokusu xenografting için Protokoller açıklanmıştır. Germ hücre nakli alıcı testiste donör kaynaklı spermatogenez ve spermatogonial kök hücreler (DGM) belirlenmesi için fonksiyonel bir sulandırma tahlil sonuçları temsil eder. Xenografting Testis dokusu alıcı farelerde testis gelişimini ve spermatogenezi çeşitli donör türleri üretir.

Germ Hücre Nakli ve Testis Dokusu Farelerde Xenografting

Germ cell transplantation was developed by Dr. Ralph Brinster and colleagues at the University of Pennsylvania in 19941,2. These ground-breaking studies ...

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29.7K Görüntüleme.  University of Calgary. Germ hücre nakli ve testis dokusu xenografting için Protokoller açıklanmıştır. Germ hücre nakli alıcı testiste donör kaynaklı spermatogenez ve spermatogonial kök hücreler (DGM) belirlenmesi için fonksiyonel bir sulandırma tahlil sonuçları temsil eder. Xenografting Testis dokusu alıcı farelerde testis gelişimini ve spermatogenezi çeşitli donör türleri üretir.

Video: Germ Cell Transplantation and Testis Tissue Xenografting in Mice

Isolation and Transplantation of Hematopoietic Stem Cells (HSCs)

Isolation and Transplantation of Hematopoietic Stem Cells HSCs

I am Christina Ceso. I am a postdoc in David's Den lab and I work on the hematopoietic stem cells. Today we are going to purify long-term recuperating hematopoietic stem cells and we'll transplant them into a mouse.We start by spraying the mice with alcohol so that first of all, the mind starts sterilized and also the hair doesn't interfere with the dissection. We use PBS with 2%serum for the dissection. I cut the back skin and I pull the skin apart in order to expose the tissue.In order to isolate the tibia, I hold the tibia with tweezers. I cut the tendon on top of the knee and I cut the muscles below the knee and after that I cut through the knee so that the TBI is not connected to the rest of the limb anymore and I pull in order to isolate it. I then hold the TB upside down and with the scissors I separate the foot from the tibia In order to obtain the femur.I cut the muscles around the femur and then I dislocate the femur from the hip. In order to isolate the hip, I cut the muscle around the hip bone on the side of the tail of the mouse and then I pull the hip away from the body of the mouth In order to isolate the spine. I cut through the tail as close as I can to the beginning of the back and then I cut on the two sides of the spine and below the spine, trying to stay as close as possible to the spine so that the peritoneum is not cut and the intestine is left untouched.A first round of cleaning of the bones is done with a scalpel, which is used to scrape the bone, the muscles away from the bone. The scalpel is good to clean the femurs, the hip and the spine. In order to clean the tibia, I hold them with forceps and I use the forceps to remove as much tissue as possible.A second round of cleaning is done using clean wipe to completely clean the bones outta the muscle, and I use the F wipe to clean the tibia, the femurs and the hips. So this is how bones look. After cleaning, I prefer 50 amount tubes with filters on top and I transfer the bones into the mortar.I add the section media and I crush the bones ask of the first round of crashing of the bones. I piped the mixture in order to release as many styles as possible into solution and reduce clumps. I transferred the mixture on the tubes through the filters and this is what the bone fragments look like after the first round of crushing.So I add more dissection medium to the bone fragments and I flash them again. I transferred the seline mixture to the tubes through the filters and at this point the remaining bone fragments are white and there is no bone marrow left. The tubes are filled with PBS 2%serum and in order to have them all with the same weight so that they're balanced in the centrifuge, but also in order to wash the filters so that we obtain as many cells as possible, I then centrifuge the cell mixture and after centrifugation, the palt includes the bone marrow cells.I aspirate the medium and I move back and forward the tube on the rack so that the pelle is becomes loose and fewer clamps are formed. When I add medium, at this point I add one ml of medium per tube and I ics since I'm sorting hematopoietic stem cells. Later on, I'm going to store an ALI output of total bone marrow sizes at this point, which I will use later for the fact single color control.I add lineage antibody cocktail. I ordered the samples to mix them well and I invaded four degrees for 15 minutes. In the meantime, I start preparing the columns for the lineage division.I put the columns on the magnet and I prepare Ian tubes under the columns. I also prepared the gas PBS using aster flip filter and attaching it to the vacuum so that all the bubbles of air move in the top part of the filter. I then add three ml of the gas DBS to each column in order for the column to become wet.After the incubation with the antibodies, I add medium to the cell samples and I centrifuge them in order to remove the excess of antibodies. After the centrifugation, the stain cells are in the pallet. I aspirate the medium and I suspend the pallet in one mile of the gas PBSI Add the strip tab beans, I mix the cells and I incubate for 15 minutes at four degrees.After the incubation, I put new elucian tubes under the columns. I add two ml of PBS to each tube with the cells and I transferred the cell mixture to the column through a 40 micron filter. I wash the tubes with more medium in order to collect all the cells remaining and I apply the remaining medium through the filters to the columns.The progenitor cells lineage low are alluded into the collection views. I pulled the luted samples and I sent tofu them. So after the centrifugation, the depleted cells are in a white pate.I aspirate the medium and re suspend the cells. I store an adequate of the depleted cells and I add the antibodies to the cell mixture for the sorting and also to all the single color controls. The antibodies are incubated in that with the size for 15 minutes in the fridge.After the ation, I add medium to the cells and and centrifuge them to remove the ex excess of antibodies. After the centrifugation, the cells are in the pt, I aspirate the media that I suspend the cells and I transfer the cells into fox tubes with a blue filter. At this point, the cells are ready to be sorted.I place, I place the mouse under a heating lamp for a few minutes so that the veins dilate and they are more easily seen. When preparing the syringe, you have to be careful to eliminate all the bubbles from the, the mixture that is in the syringe and then you bend the needle at 90 degrees. I put the mouse under a glass cup with a weight on top so that it can run away.I sterilize the tail with the alcohol pad so that the site of injection is sterile, but also it's easier to identify the vein. If you look at the tail of the mouse from the top in the middle and top of the tail, there is an artery while on the two sides there are the two veins and we inject the cells in one of the two veins. I insert the needle into the vein and I inject following injection.It is to wait for a couple of seconds before removing the needle, the needle, and after that I clean the side of injection. This is a representation of the syringe with the ben needle bent at 90 degrees with the knee, the at this ankle compared to the tail, which makes the injection the easiest. When you injecting the tail of the mouse, you have to keep in mind that the tail vein is extremely superficial, so really this is the orientation of the needle compared to the tail.And it looks like you will just keep sliding on top of the tail, but actually you will get into the tail then. I am Christina Ceso. I am a post in David Kaden lab, and I work on the hematopoietic stem cells.I usually use for animals to start with, and I had more variability at the beginning, but once you become consistent with your methods, I consistently get about 50, 000 to 60, 000 stem cells out for mice. I did my PhD in London with Dr.Fiona Wat working on derma stem cells. After that, I just wanted to have a wider knowledge of adult stem cells in various tissues, and so I decided to move to David Kaden lab to work on hematopoietic stem cells, mainly because the things that are known about hematopoietic stem cells are the things that are not known for the epidermic stem cells and a number of assays that can be done with epi.Epidermal stem cells are not really done yet with the hematopoietic stem cells, and so at this point I'm trying to put together the, the knowledge from the two different fields and ultimately I'm interested in the study of the mechanism to regulate adult tissue sensors in general, so not just the epidermis or the stem cells, but also different tissues because there are, there are similar mechanism that is, it's always the same genes that are involved in the regulation that just interact with each other in different ways, and it's just interesting to understand what makes each stem cell be a stem, but then ultimately a stem cell in a different tissue. So giving rise to a different protein.

İzolasyon ve Hematopoetik Kök Hücre Transplantasyonu (HKH&#39;lerin)

Biyoloji

Heterotopic Heart Transplantation in Mice

The mouse heterotopic heart transplantation has been used widely since it was introduced by Drs. Corry and Russell in 1973. It is particularly valuable for studying rejection and immune response now that newer transgenic and gene knockout mice are available, and a large number of immunologic reagents have been developed. The heart transplant model is less stringent than the skin transplant models, although technically more challenging. We have developed a modified technique and have completed over 1000 successful cases of heterotopic heart transplantation in mice. When making anastomosis of the ascending aorta and abdominal aorta, two stay sutures are placed at the proximal and distal apexes of recipient abdominal aorta with the donor s ascending aorta, then using 11-0 suture for anastomosis on both side of aorta with continuing sutures. The stay sutures make the anastomosis easier and 11-0 is an ideal suture size to avoid bleeding and thrombosis.
When making anastomosis of pulmonary artery and inferior vena cava, two stay sutures are made at the proximal apex and distal apex of the recipient s inferior vena cava with the donor s pulmonary artery. The left wall of the inferior vena cava and donor s pulmonary artery is closed with continuing sutures in the inside of the inferior vena cava after, one knot with the proximal apex stay suture the right wall of the inferior vena cava and the donor s pulmonary artery are closed with continuing sutures outside the inferior vena cave with 10-0 sutures. This method is easier to perform because anastomosis is made just on the one side of the inferior vena cava and 10-0 sutures is the right size to avoid bleeding and thrombosis. In this article, we provide details of the technique to supplement the video.

The mouse heterotopic heart transplantation model has been proven by many investigators to be an important method for studying mechanisms of rejection and immune response. However, the techniques involved are still challenging. By modifying standard techniques we have had success with more than 1000 transplants.

Farelerde Heterotopik Kalp Nakli

The mouse heterotopic heart transplantation has been used widely since it was introduced by Drs. Corry and Russell in 1973. It is particularly valuable ...

Small Bowel Transplantation In Mice

Since 1990, the development of tacrolimus-based immunosuppression and improved surgical techniques, the increased array of potent immunosuppressive medications, infection prophylaxis, and suitable patient selection helped improve actuarial graft and patient survival rates for all types of intestine transplantation. Patients with irreversible intestinal failure and complications of parenteral nutrition should now be routinely considered for small intestine transplantation. However, Survival rates for small intestinal transplantation have been slow to improve compares increasingly favorably with renal, liver, heart and lung. The small bowel transplantation is still unsatisfactory compared with other organs. Further progress may depend on better understanding of immunology and physiology of the graft and can be greatly facilitated by animal models. A wider use of mouse small bowel transplantation model is needed in the study of immunology and physiology of the transplantation gut as well as efficient methods in diagnosing early rejection. However, this model is limited to use because the techniques involved is an extremely technically challenging. We have developed a modified technique. When making anastomosis of portal vein and inferior vena cava, two stay sutures are made at the proximal apex and distal apex of the recipient s inferior vena cava with the donor s portal vein. The left wall of the inferior vena cava and donor s portal vein is closed with continuing sutures in the inside of the inferior vena cava after, after one knot with the proximal apex stay suture the right wall of the inferior vena cava and the donor s portal vein are closed with continuing sutures outside the inferior vena cave with 10-0 sutures. This method is easier to perform because anastomosis is made just on the one side of the inferior vena cava and 10-0 sutures is the right size to avoid bleeding and thrombosis.  In this article, we provide details of the technique to supplement the video.

The mouse small bowel transplantation model has been recognized as an important tool to study mechanismes of immune rejection and screen new immunosuppressive drugs. However, this model is limited to use because the techniques involved is an extremely technically challenge. Now we introduce the modified technique.

Farelerde Küçük Bağırsak Nakli

Since 1990, the development of tacrolimus-based immunosuppression and improved surgical techniques, the increased array of potent immunosuppressive ...

Actin Co-Sedimentation Assay; for the Analysis of Protein Binding to F-Actin

The actin cytoskeleton within the cell is a network of actin filaments that allows the movement of cells and cellular processes, and that generates tension and helps maintains cellular shape.  Although the actin cytoskeleton is a rigid structure, it is a dynamic structure that is constantly remodeling.  A number of proteins can bind to the actin cytoskeleton.  The binding of a particular protein to F-actin is often desired to support cell biological observations or to further understand dynamic processes due to remodeling of the actin cytoskeleton. The actin co-sedimentation assay is an in vitro assay routinely used to analyze the binding of specific proteins or protein domains with F-actin.  The basic principles of the assay involve an incubation of the protein of interest (full length or domain of) with F-actin, ultracentrifugation step to pellet F-actin and analysis of the protein co-sedimenting with F-actin.  Actin co-sedimentation assays can be designed accordingly to measure actin binding affinities and in competition assays.

Proteins bind to filamentous actin (F-actin) through distinct actin binding modules. In this video we demonstrate the procedure of actin co-sedimentation, which is an in vitro assay routinely used to analyze proteins or specific domains that bind F-actin.

Aktin Co-Sedimantasyon Testi; F-Aktin Bağlayıcı Protein Analizi

The actin cytoskeleton within the cell is a network of actin filaments that allows the movement of cells and cellular processes, and that generates ...

Generation of Human CD40-activated B cells

CD40-activated B cells (CD40-B cells) have been identified as an alternative source of immuno-stimulatory antigen-presenting cells (APC) for cancer immunotherapy 1-3. Compared to Dendritic cells (DCs), the best characterized APC, CD40-B cells have several distinct biological and technical properties. Similar to DCs, B cells show an increased expression of MHC and co-stimulatory molecules (Fig.1b), exhibit a strong migratory capacity and present antigen presentation efficiently to T cells, after stimulation with interleukin-4 and CD40 ligand (CD40L). However, in contrast to immature or mature DCs, CD40-B cells express the full lymph node homing triad consisting of CD62L, CCR7/CXCR4, and leukocyte function antigen-1 (LFA1, CD11a/CD18), necessary for homing to secondary lymphoid organs (Fig.1a) 3. CD40-B cells can be generated without difficulties from very small amounts of peripheral blood which can be further expanded in vitro to very large amounts of highly-pure CD40-B cells (&gt;109 cells per patient) from healthy donors as well as cancer patients (Fig.1c,d) 1,4. 
In this protocol we demonstrate how to obtain fully activated CD40-B cells from human PBMC. Key molecules for the cell culture are CD40 ligand, interleukin-4 (IL-4) and cyclosporin A (CsA), which are replenished in a 3-4 day culture cycle. For laboratory purposes CD40-stimulation is provided by NIH/3T3 cells expressing recombinant human CD40 ligand (tCD40L NIH/3T3) 5. To avoid contamination with non-transfected cells, expression of the human CD40 ligand on the transfectants has to be checked regularly (Fig.2). 
After 14 days CD40-B cell cultures consist of more than 95% pure B cells and an expansion of CD40-B cells over 65 days is frequently possible without any loss of function 1, 4. CD40-B cells efficiently take up, process and present antigens to T cells 6. They do not only prime na&#970;ve, but also expand memory T cells 7,8. CD40-activated B cells can be used to study B-cell activation, differentiation and function. Moreover, they represent a promising tool for therapeutic or preventive vaccination against tumors 9.

In this video we present the ex vivo generation and expansion of human CD40-activated B cells (CD40-B) from peripheral blood mononuclear cells (PBMC) by stimulation with CD40 ligand and interleukin-4.

İnsan CD40 aktive B hücreleri üretilmesi

CD40-activated B cells (CD40-B cells) have been identified as an alternative source of immuno-stimulatory antigen-presenting cells (APC) for cancer ...

Tissue Targeted Embryonic Chimeras: Zebrafish Gastrula Cell Transplantation

Certain fundamental questions in the field of developmental biology can only be answered when cells are placed in novel environments or when small groups of cells in a larger context are altered.  Watching how one cell interacts with and behaves in a unique environment is essential to characterizing cell functions.  Determining how the localized misexpression of a specific protein influences surrounding cells provides insightful information on the roles that protein plays in a variety of developmental processes.  Our lab uses the zebrafish model system to uniquely combine genetic approaches with classical transplantation techniques to generate genotypic or phenotypic chimeras.  We study neuron-glial cell interactions during the formation of forebrain commissures in zebrafish. This video describes a method that allows our lab to investigate the role of astroglial populations in the diencephalon and the roles of specific guidance cues that influence projecting axons as they cross the midline. Due to their transparency zebrafish embryos are ideal models for this type of ectopic cell placement or localized gene misexpression. Tracking transplanted cells can be accomplished using a vital dye or a transgenic fish line expressing a fluorescent protein. We demonstrate here how to prepare donor embryos with a vital dye tracer for transplantation, as well as how to extract and transplant cells from one gastrula staged embryo to another.  We present data showing ectopic GFP+ transgenic cells within the forebrain of zebrafish embryos and characterize the location of these cells with respect to forebrain commissures.  In addition, we show laser scanning confocal timelapse microscopy of Alexa 594 labeled cells transplanted into a GFP+ transgenic host embryo.  These data provide evidence that gastrula staged transplantation enables the targeted positioning of ectopic cells to address a variety of questions in Developmental Biology.

Zebrafish cell transplantation enables the combination of genetics and embryology to generate tissue specific chimeras. This video demonstrates gastrula staged cell transplantations that have allowed our lab to investigate the roles of astroglial populations and specific guidance cues during commissure formation in the forebrain.

Doku Hedeflenen Embriyonik Chimeras: Zebra balığı Gastrula Hücre Nakli

Certain fundamental questions in the field of developmental biology can only be answered when cells are placed in novel environments or when small groups ...

Heterotopic and Orthotopic Tracheal Transplantation in Mice used as Models to Study the Development of  Obliterative Airway Disease

Obliterative airway disease (OAD) is the major complication after lung transplantations that limits long term survival (1-7).
To study the pathophysiology, treatment and prevention of OAD, different animal models of tracheal transplantation in rodents have been developed (1-7). Here, we use two established models of trachea transplantation, the heterotopic and orthotopic model and demonstrate their advantages and limitations.
For the heterotopic model, the donor trachea is wrapped into the greater omentum of the recipient, whereas the donor trachea is anastomosed by end-to-end anastomosis in the orthotopic model.
In both models, the development of obliterative lesions histological similar to clinical OAD has been demonstrated (1-7).
This video shows how to perform both, the heterotopic as well as the orthotopic tracheal transplantation technique in mice, and compares the time course of OAD development in both models using histology.

This video shows and compares two experimental models to study the development of obliterative airway disease (OAD) in mice, the heterotopic and orthotopic tracheal transplantation model.

Obliteratif Havayolu Hastalığı Geliştirme Eğitim Modeli olarak kullanılan Farelerde Heterotopik ve Ortotopik Trakeal Nakli

Obliterative airway disease (OAD) is the major complication after lung transplantations that limits long term survival (1-7).
To study the ...

Planarian Immobilization, Partial Irradiation, and Tissue Transplantation

The planarian, a freshwater flatworm, has proven to be a powerful system for dissecting metazoan regeneration and stem cell biology1,2. Planarian regeneration of any missing or damaged tissues is made possible by adult stem cells termed neoblasts3. Although these stem cells have been definitively shown to be pluripotent and singularly capable of reconstituting an entire animal4, the heterogeneity within the stem cell population and the dynamics of their cellular behaviors remain largely unresolved. Due to the large number and wide distribution of stem cells throughout the planarian body plan, advanced methods for manipulating subpopulations of stem cells for molecular and functional study in vivo are needed.
Tissue transplantation and partial irradiation are two methods by which a subpopulation of planarian stem cells can be isolated for further study. Each technique has distinct advantages. Tissue transplantation allows for the introduction of stem cells, into a na&iuml;ve host, that are either inherently genetically distinct or have been previously treated pharmacologically. Alternatively, partial irradiation allows for the isolation of stem cells within a host, juxtaposed to tissue devoid of stem cells, without the introduction of a wound or any breech in tissue integrity. Using these two methods, one can investigate the cell autonomous and non-autonomous factors that control stem cell functions, such as proliferation, differentiation, and migration.
Both tissue transplantation5,6 and partial irradiation7 have been used historically in defining many of the questions about planarian regeneration that remain under study today. However, these techniques have remained underused due to the laborious and inconsistent nature of previous methods. The protocols presented here represent a large step forward in decreasing the time and effort necessary to reproducibly generate large numbers of grafted or partially irradiated animals with efficacies approaching 100 percent. We cover the culture of large animals, immobilization, preparation for partial irradiation, tissue transplantation, and the optimization of animal recovery. Furthermore, the work described here demonstrates the first application of the partial irradiation method for use with the most widely studied planarian, Schmidtea mediterranea. Additionally, efficient tissue grafting in planaria opens the door for the functional testing of subpopulations of na&iuml;ve or treated stem cells in repopulation assays, which has long been the gold-standard method of assaying adult stem cell potential in mammals8. Broad adoption of these techniques will no doubt lead to a better understanding of the cellular behaviors of adult stem cells during tissue homeostasis and regeneration.

An effective method for grafting tissue of defined and consistent size between planaria is described. Also included is a description of how the immobilization technique used for transplantation can be adapted, in conjunction with lead shields, for the partial irradiation of live animals.

Planarian İmmobilizasyon, Kısmi Işınlama, ve Doku Nakli

The planarian, a freshwater flatworm, has proven to be a powerful system for dissecting metazoan regeneration and stem cell biology1,2. Planarian ...

Studying Proteolysis of Cyclin B at the Single Cell Level in Whole Cell Populations

Equal distribution of chromosomes between the two daughter cells during cell division is a prerequisite for guaranteeing genetic stability 1. Inaccuracies during chromosome separation are a hallmark of malignancy and associated with progressive disease 2-4. The spindle assembly checkpoint (SAC) is a mitotic surveillance mechanism that holds back cells at metaphase until every single chromosome has established a stable bipolar attachment to the mitotic spindle1. The SAC exerts its function by interference with the activating APC/C subunit Cdc20 to block proteolysis of securin and cyclin B and thus chromosome separation and mitotic exit. Improper attachment of chromosomes prevents silencing of SAC signaling and causes continued inhibition of APC/CCdc20 until the problem is solved to avoid chromosome missegregation, aneuploidy and malignant growths1.
Most studies that addressed the influence of improper chromosomal attachment on APC/C-dependent proteolysis took advantage of spindle disruption using depolymerizing or microtubule-stabilizing drugs to interfere with chromosomal attachment to microtubules. Since interference with microtubule kinetics can affect the transport and localization of critical regulators, these procedures bear a risk of inducing artificial effects 5.
To study how the SAC interferes with APC/C-dependent proteolysis of cyclin B during mitosis in unperturbed cell populations, we established a histone H2-GFP-based system which allowed the simultaneous monitoring of metaphase alignment of mitotic chromosomes and proteolysis of cyclin B 6.
To depict proteolytic profiles, we generated a chimeric cyclin B reporter molecule with a C-terminal SNAP moiety 6 (Figure 1). In a self-labeling reaction, the SNAP-moiety is able to form covalent bonds with alkylguanine-carriers (SNAP substrate) 7,8 (Figure 1). SNAP substrate molecules are readily available and carry a broad spectrum of different fluorochromes. Chimeric cyclin B-SNAP molecules become labeled upon addition of the membrane-permeable SNAP substrate to the growth medium 7 (Figure 1). Following the labeling reaction, the cyclin B-SNAP fluorescence intensity drops in a pulse-chase reaction-like manner and fluorescence intensities reflect levels of cyclin B degradation 6 (Figure 1). Our system facilitates the monitoring of mitotic APC/C-dependent proteolysis in large numbers of cells (or several cell populations) in parallel. Thereby, the system may be a valuable tool to identify agents/small molecules that are able to interfere with proteolytic activity at the metaphase to anaphase transition. Moreover, as synthesis of cyclin B during mitosis has recently been suggested as an important mechanism in fostering a mitotic block in mice and humans by keeping cyclin B expression levels stable 9,10, this system enabled us to analyze cyclin B proteolysis as one element of a balanced equilibrium 6.

Metaphase to anaphase transition is triggered through anaphase-promoting complex (APC/C)-dependent ubiquitination and subsequent destruction of cyclin B. Here, we established a system which, following pulse-chase labeling, allows monitoring cyclin B proteolysis in entire cell populations and facilitates the detection of interference by the mitotic checkpoint.

Tüm Cep Populasyonlarının Tek Hücre Düzeyinde Siklin B Proteoliz incelenmesi

Equal distribution of chromosomes between the two daughter cells during cell division is a prerequisite for guaranteeing genetic stability 1. Inaccuracies ...

Isolation, Culture, and Transplantation of Muscle Satellite Cells

Muscle satellite cells are a stem cell population required for postnatal skeletal muscle development and regeneration, accounting for 2-5% of sublaminal nuclei in muscle fibers. In adult muscle, satellite cells are normally mitotically quiescent. Following injury, however, satellite cells initiate cellular proliferation to produce myoblasts, their progenies, to mediate the regeneration of muscle. Transplantation of satellite cell-derived myoblasts has been widely studied as a possible therapy for several regenerative diseases including muscular dystrophy, heart failure, and urological dysfunction. Myoblast transplantation into dystrophic skeletal muscle, infarcted heart, and dysfunctioning urinary ducts has shown that engrafted myoblasts can differentiate into muscle fibers in the host tissues and display partial functional improvement in these diseases. Therefore, the development of efficient purification methods of quiescent satellite cells from skeletal muscle, as well as the establishment of satellite cell-derived myoblast cultures and transplantation methods for myoblasts, are essential for understanding the molecular mechanisms behind satellite cell self-renewal, activation, and differentiation. Additionally, the development of cell-based therapies for muscular dystrophy and other regenerative diseases are also dependent upon these factors.

However, current prospective purification methods of quiescent satellite cells require the use of expensive fluorescence-activated cell sorting (FACS) machines. Here, we present a new method for the rapid, economical, and reliable purification of quiescent satellite cells from adult mouse skeletal muscle by enzymatic dissociation followed by magnetic-activated cell sorting (MACS). Following isolation of pure quiescent satellite cells, these cells can be cultured to obtain large numbers of myoblasts after several passages. These freshly isolated quiescent satellite cells or ex vivo expanded myoblasts can be transplanted into cardiotoxin (CTX)-induced regenerating mouse skeletal muscle to examine the contribution of donor-derived cells to regenerating muscle fibers, as well as to satellite cell compartments for the examination of self-renewal activities.

The isolation and culture of a pure population of quiescent satellite cells, a muscle stem cell population, is essential to the understanding of muscle stem cell biology and regeneration, as well as stem cell transplantation for therapies in muscular dystrophy and other degenerative diseases.&#160;

Kas Uydu Hücreleri İzolasyon, Kültür ve Transplantasyon

Muscle satellite cells are a stem cell population required for postnatal skeletal muscle development and regeneration, accounting for 2-5% of sublaminal ...