Mutant worm strains were kindly provided by the Caenorhabditis Genetics Center. This research was supported by a grant of the Korea Health Technology R&amp;D Project through the Korea Health Industry Development Institute (KHIDI), funded by the Ministry of Health &amp; Welfare, Republic of Korea (grant number: HI14C1277).

PCR ile, Digoksigenin-dUTP ile 1. etiketleme probları <ol><li> Daha önce tarif edildiği gibi 13 10x dNTP karışımı digoksijenin-dUTP ihtiva eden PCR etiketlenmesi. </li><li> üreticinin talimatlarına uygun olarak yan kolon kromatografisi ile PCR ürünü saflaştırılır. <ol><li> Prob daha kısa 200 bp takdirde, reaksiyon karışımından yan sütun kromatografisi yerine spin kolon saflaştırılması serbest digoksijenin-dUTP çıkarın. </li></ol></li></ol> 2. Hazırlama Polilisin Kaplı Slaytlar Not: Tüm işlem yaklaşık 2 saat sürer. adımların çoğu kurutma adımı hariç, oda sıcaklığında gerçekleştirilir. <ol><li> slaytlar temizlik <ol><li> plastik bir kap slaytlar yerleştirin ve kısaca damıtılmış su (DW) ile slaytlar durulayın. suyu çıkarmak ve DW% 1 cam temizleyici içeren kabı doldurun. </li><li> Oda sıcaklığında 50 rpm&#39;de (RT) 15 dakika boyunca slaytlar çalkalayın. DW 3 slaytlar yıkayınOda sıcaklığında her biri 5 dakika için süre. </li><li> çalkalama ile 15 dakika boyunca% 70 etanol ile slaytlar yıkayın. % 70 etanol atın ve 15 dakika için 65 ° C kuru blok ve kuru hava slaytlar yerleştirin. </li></ol></li><li> Çok iyi cam slaytların polilisin kaplama Not: boyama işlemi boyunca örnek yapışma sağlar çünkü slayt cam Polilisin kaplama, önemli bir adımdır. Kötü kaplanmış slayt örnek kaybına neden olacaktır. <ol><li> % 0.01 polilisin stok solüsyonu seyreltik (a / h), distile su içinde. seyreltilmiş,% 0.01 20 ul temiz bir cam slayt oyuklarına (ağırlık / hacim) polilisin ekleyin. </li><li> Oda sıcaklığında 5 dakika boyunca cam slayt inkübe edin. </li><li> 1 saat için 65 ° C kuru blok ve kuru hava slaytlar yerleştirin. </li><li> tozsuz kutuya polilisin slaytlar saklayın. </li></ol></li></ol> Slayt Camına Worms 3. Sabitleme (Şekil 1) <ol><li> FISH için hazırlanıyor embriyolar Not: Hasat solucanlar tüm t önceO bakteriyel gıda mikroskop altında büyüme ortamı izleyerek tüketilmektedir. Açlık erişkin solucanlar yumurta üretimini azaltır ve yumurta taramayı artırır. Ayrıntılı yöntemler, 14,15 tarif edilmiştir. <ol><li> Standart yöntemlere 14 göre 50 mm Petri-çanak solucanlar büyütün. </li><li> tüm bakteriyel gıda tüketilen sonra spatula ile çeyrek agar ortamı kesti. bulaşmasını önlemek için kesmeden önce spatula sterilize edin. </li><li> 100 mm nematod büyüme ortamı (NGM) plaka üzerinde agar tüm parçası koyun. Taze bakteriyel gıda ulaşmak için solucanlar için baş aşağı agar parça çevirin. </li><li> 48 saat 72 sonra M9 tamponu ile solucanlar toplamak. NGM plaka üzerinde M9 tampon 5 ml - 3 ekleyin. NGM yüzeyinde Pipet M9 tampon solucanlar yıkayın. </li><li> solucanlar sıvıyı toplamak ve 15 ml&#39;lik bir tüpe ilave edin. Not:% 30 sukroz çözeltisi içinde hasat santrifüj solucanlar sonra agar bir pislik değilse. kalıntı pellet haline iken, solucanlar üzerinde yüzeryüzey. </li><li> 15 ml hacmi telafi etmek M9 tampon ekleyin. 3 dakika boyunca 300 x g&#39;de santrifüj ile solucanlar pelet ve M9 tampon en çıkarmak. Bu adımı 2 kez daha tekrarlayın. Not: Solucanlar hala santrifüj sonra yüzen ise, santrifüj fren seviyesi = 1 değerine ayarlayın. </li><li> Aspire M9 tampon. solucanlar ağartma çözüm ekleyin. solucanlar 0.5 ml başına 5 M KOH 6,5 DW ml, hipoklorit 2 mi, 1 ml ilave edilir. </li><li> 50 rpm&#39;de 3 dakika boyunca oda sıcaklığında çalkalanarak solucanlar inkübe edin. 15 saniye vorteks solucanlar mekanik solucanlar kesme ve yumurta ortaya çıkarmak. </li><li> beyazlatma sırasında bir diseksiyon mikroskobu altında tüp gözlemleyin. solucanlar yarıda kesilir ve yumurta serbest olduğundan emin olun. Yetişkin vücudunda en çözülür, 15 ml hacim oluşturan M9 tampon eklemek. </li><li> 3 dakika boyunca 300 x g&#39;de santrifüjleyin. Aspire M9 en ve 15 ml hacmi telafi etmek için taze M9 tampon ekleyin. Tekrarlayın yıkama adımı 3 kez. Not:Onlar ağartma sürecini engelleyen olarak, solucanlar aşırı miktarda kullanmaktan kaçının. az 8 dk yıkama kadar toplam reaksiyon süresini tutun. Aşırı ağartılmış yumurta güçlü otofloresans üretir. </li><li> 200 ul% 2 PFA yapmak için% 4 paraformaldehid (PFA), 200 ul kadar polisorbat-20 (PBST) ile fosfat tamponlu tuzlu su ekleyin. Dikkat: PFA kanserojen olduğundan, PFA kullanmadan önce koruyucu giysi, eldiven ve göz maskesi kullanınız. </li><li> polilizin kaplı slayt kuyusuna üzerine% 2 PFA yumurta 40 ul ekleyin. </li><li> nemli bir oda içinde slaytlar yerleştirin ve oda sıcaklığında 15 dakika inkübe edilir. slaytlar içine yerleştirilir hemen sonra nemli odasına kapatın. Not: Sabit olurken yumurta slayt altına yerleşmek. </li></ol></li><li> FISH için disseke gonadlar hazırlanması <ol><li> Hasat yetişkin kurtlar M9 1 ml tampon pipetleme 50 mm NGM plaka üzerinde büyütülür. Bakteriyel gıda önce hasat solucanlar tükenmiş. </li><li> herhangi bir bakteri zekâ solucanlar yıkayınh M9 tampon, 2 kez. Not: Artık bakteri polilisin kaplı slaytlar yapışmasını disseke gonadlar ile etkileşime girebilir. </li><li> 300 x g santrifüj ile solucanlar Pelet. M9 çıkarın ve mikropipet ile boş NGM plaka solucanlar aktarın. </li><li> polilisin tedavi slayt bir kuyunun 2 mM levamizol içeren M9 tampon 30 ul ekleyin. </li><li> 1 ml tüp M9 tampon 500 ul ekle. ağız pipet ile solucanlar aktarmak için bu tampon kullanın. </li><li> M9 tampon içeren 1 ml tüp bir kılcal koyarak M9 tamponu ile ağız pipet ucu doldurun. </li><li> Solucanların kafa ağız pipet girer, böylece diseksiyon mikroskobu altında, sadece yetişkin solucan ve sürükleme ağız pipet başın önünde ağız pipet ucu koydu. Solucanların baş ucu girdiğinde, solucanın tüm vücut ucu içine çekilecektir. </li><li> ağız pipet kullanarak polilisin kaplı slayt solucanlar aktarın. </li><li> kesmek, bir jilet kullanarakkafa veya slaytta solucanlar kuyruk ucu f. solucan kesildiğinde, gonadlar dışarı çıkacaktır. Gonadlar slaytlar sopa olacak. <ol><li> bir kuyuda en az 30 solucanlar hazırlayın. Daha fazla kuyu bir 30 solucanlar için kullanılabilir. </li></ol></li><li> Nemli odasında slayt koymak ve ağız pipet ile M9 tampon aspire. </li><li> nemli odada 15 dakika boyunca oda sıcaklığında% 2 PFA 20 ul ekleyerek örnek sabitleyin. </li></ol></li></ol> 4. Sabitleme ve Permeabilization <ol><li> kuru buz üzerinde bir alüminyum blok yerleştirin ve derin dondurucuda saklayın (-80 ° C). -20 ° C&#39;de saklayın, metanol ve aseton. </li><li> PFA fiksasyon adımı 3.1.15 veya 3.2.11 sonra ~ sabitleştirici 5 ul mikropipet bırakarak kullanarak fiksatif kaldırın. </li><li> Örnek slaytta başka polilisin kaplı slayt koyun. çözüm aşırı ise kağıt havluyla Fiksatif çıkarın. Onlar araya sıkışmış bir kez slaytlar hareket ettirmeyin. </li><li> slaytlar FreezeEn az 15 dakika süreyle alüminyum bloğunun üzerine. Not: Örnekler, en az 2 saklanabilir - 3 gün. </li><li> slaytlar dondurulmuş edilirken, buz üzerinde soğuk metanol ve aseton içeren kavanoz koydu. </li><li> slaytlar almak ve örnek dondurarak çatlamak bükün. Üst slayt atın. Hemen 5 dakika boyunca buz soğukluğunda metanol içine slayt ıslatın. </li><li> 5 dakika boyunca buz soğukluğunda aseton slaytlar aktarın. </li><li> Kalıntı fiksatif kaldırmak için 5 dakika boyunca slaytlar PBST ile 3 kez yıkayın. Bir sonraki adıma geçin ya da 4 ° C&#39;de% 100 etanol slaytlar saklayın. Not: Örnekler, en az 2 saklanabilir - 3 gün. </li></ol> Sabit Hücre 5. Melezleme <ol><li> (PBST 10 ug / ml RNase A ihtiva eden) RNaz çözeltisi 20 ul ekle. 1 saat boyunca 37 ° C&#39;de nemli bir oda içinde slayt inkübe edin. </li><li> polisorbat-20 (2x SSCT) her biri 15 dakika için 2x tuzlu su ve sodyum sitrat iki slayt yıkayın. </li><li> 20 eklemelezleştirme çözeltisi ul ve 37 ° C inkübatör nemli odasına koyun. 1 saat sonra, pipetleme hibridizasyon çözeltisi çıkarın. </li><li> hibridizasyon çözüm çıkarmadan önce, probu hazırlamak. Prob, çift sarmallı DNA&#39;dır, kuru bir blok üzerinde 5 dakika boyunca 95 ° C&#39;de ısıtılarak probları denatüre. ısıtıldıktan sonra, buz kısaca probu serin. </li><li> örnek probları içeren hibridizasyon çözeltisi 10 ul ekle. 2,000 DIG etiketli prob için, 1 bir oranda kullanım kesafeti: 200 PNA probu, 1 oranında kullanımı konsantrasyonu için. cam kapak ile örnek örtün. </li><li> ısı bloğu (80 ° C) su ile ıslatılmış kağıt havlu koyun. nem ve sıcaklık korumak için ısı bloğu üzerinde plastik bir kutu kapağını takın. </li><li> ısıtma bloğu sıcaklığı (80 ° C) stabil hale sonra ısıtılmış bir kağıt havlu üzerine Örnek slayt yerleştirin ve plastik kutu kapağı örnekleri kapsamaktadır. 3 dakika boyunca numune denatüre. </li><li> incgece boyunca 37 ° C&#39;de nemli bir oda içinde slaytlar Ubate. </li></ol> 6. yıkar ve İmmünofloresan <ol><li> 37 ° C hibridizasyon yıkama solüsyonu (2X SSC,% 50 formamid) ısıtın. </li><li> 5 dakika boyunca oda sıcaklığında iki kez PBST içinde örnek yıkayın. cam kapak çıkarın. </li><li> 30 dakika boyunca 37 ° C&#39;de hibridizasyon yıkama çözeltisi içinde örnek yıkayın. </li><li> 3 kez oda sıcaklığında PBST içinde Örnek slayt yıkayın. Not: oda sıcaklığında nemli odasında sonraki tüm adımları uygulayın. </li><li> Bloke etme çözeltisinin 20 ul ekle ve nemli oda içinde oda sıcaklığında 1 saat inkübe edin. </li><li> bloke etme çözeltisi çıkarın ve FITC ile birleşik anti-digoksigenin antikor çözeltisi (1: 200) ekleyin, oda sıcaklığında ya da gece boyunca 4 ° C&#39;de 3 saat karıştırıldı. </li></ol> 7. Montaj ve Gözlem <ol><li> 15 dakika her biri için PBST ile 2 kez örnek slayt yıkayın. </li><li> DAPI ile montaj çözümü 10 ul ekle. nazikçe cam kapak ve basın koyun. herhangi bir fazla çözeltiyiBir kağıt havlu ile. </li><li> montaj çözümü buharlaşmasını önlemek için, oje ile kapak cam kenarları mühür. </li><li> konfokal mikroskop altında uyun. yüksek arka plan ile embriyolar hariç. 20 çekirdekleri - 4 sahip bir alan üzerinde odaklanın. </li><li> 100X objektif lens ile üreticinin talimatlarına göre fotoğraf çekmek. Not: FITC için 488 nm lazer ile Cy3 için 555 nm lazer ile, DAPI için 405 nm lazer ile örnek Excite. </li></ol> Telomer Sinyal 8. sayısallaştırılması Not: Daha önce 16 açıklandığı gibi Kantitasyonu yapıldı. karşılaştırılacak olan tüm resimler maruz kalma süresi ve ışık kaynağı içeren aynı ortamda alınmalıdır. <ol><li> tif formatında görüntü vermek. </li><li> Indirin ve görüntü analiz yazılımı yükleyin. </li><li> Görüntü analiz yazılımı çalıştırın ve düğmeyi katılıyorum tıklayın. </li><li> Açık düğmesine tıklayın. görüntü dosyasını çift tıklatarak telomer FISH ile görüntüleri açın. </li><li> tıklayın[Değiştir] - [select işleme bölge], sol-tıklayın ve sürükleyerek ilgi seçin bölgesi. Non-spesifik boyama hariç. </li><li> [Optik yoğunlukları nokta] telomer sinyali ile kanalı seçin ve .txt dosyasına sonuçları kaydetmek için dosya adını girin - [tedbiri] tıklayın. Not: sonuçların sütun aşağıdaki sırayla şunlardır: spot Floresan, spot ve nokta alanının arka plan yoğunluğu. </li><li> değerleri kopyalamak ve spot floresan gelen spot arka plan yoğunluğu çıkarın. değerleri artık istatistiksel analiz edilebilir. </li></ol>

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The authors have nothing to disclose.

Protokolde ana avantajı, hücresel yapının morfolojisine fark zarar vermeden prosedür basitliğidir. Birkaç adım C için optimize edildi Bu protokolde elegans BALIK. Başarılı FISH için kritik adımlar probların etiketleme, embriyo ve penetrasyon tespit sayılabilir. Digoksigenin dUTP etiketleme yöntemi PCR ya da nik-çevirisi ile kolay kullanımlı etiketleme yöntemi temin etmektedir. Uzun hedef dizisi etiketlemek için, nick-çeviri tercih edilir. Bu durumda, sondalar sondaların sızmas?...

Telomer olarak anormal füzyon ve bozulma kromozomal uçları korur. Memeli telomer G bakımından zengin heksamerik tekrarlar, TTAGGG ve shelterin kompleksleri oluşur. nematodun telomer tekrar dizisi memelilerde (TTAGGC) benzer olan. Çoğu ökaryotlar kendi kromozom uçları telomer tekrarlar eklemek için telomeraz kullanmaktadır. Ancak, 10 - kanser hücrelerinin% 15 Telomerlerin (ALT) 3 Alternatif uzatma olarak bilinen telomeraz bağımsız bir mekanizma kullanmaktadır. Daha önce, telomer tekrarlar ve tAlt olarak adlandırılan ilişkili diziler, kritik sterilite 2 hayatta telomeraz mutant soylarının telomerlerin amplifiye bildirmiştir. Telomer uzunluğu kantitatif PCR veya toplam telomerlerin 4,5,6,7 ortalama uzunluğu içerir Southern blot ile ölçülmüştür. Tüm genom dizileme verileri telomer tekrarın Oku sayısı da toplam telomer içeriğinin 8 bir göstergesidir. Sin rağmengle telomer uzunluğunun Analizi (STELA) tek bir telomer uzunluğu, bu telomerlerin 9 mekansal bilgi sağlayamaz sağlayabilir. POT-1 :: MCherry muhabiri protein in vivo telomerlerin mekansal bilgi sağlarken POT-1 proteini 10 bağlayıcı tek iplikli telomer olduğu gibi, bu çift sarmallı telomer uzunluklarını temsil edemez. Yukarıda bahsedilen yöntemler dizilerin ortalama bilgi sağlar birlikte, in situ hibridizasyon (FISH) floresan kromozomal ölçekte miktarı ve ilgi duyulan tek tek dizilerin uzamsal desen gözlemlenebilir. Bunun yerine DNA saflaştırma, doku ya da hücre FISH nativ uzamsal bilgiler korumak için sabitlenir. Böylece, BALIK gibi telomer tekrarlar gibi bireysel tekrar dizilerinin, gözlem için bir nicel ve nitel bir araçtır. Bu protokol, hem Telo eş zamanlı saptanması için etkili bir yöntem sağlarsadece ve diğer tekrarlar daha önce açıklanan yöntemlerden 11,12 den iyileştirmeler dayalı. C. elegans larvaları veya yetişkin yüksek farklılaşmış hücreler ile çok hücreli organizma bulunmaktadır. hücrelerin heterojen telomer noktalar sayıda kantitatif analizi engellemektedir. analiz hücrelerin sayısını arttırmak için, embriyolar izole ve FISH için polilisin ile kaplanmış dilimlerin üzerine yayılırlar. Buna ek olarak, bu protokol, aynı zamanda, bağışıklık ile kombine edilebilir. protokol çalışan bir kanıtı olarak, biz gözlemlemek ve iki farklı tekrarlayan dizileri ölçmek mümkün olduğunu göstermektedir. TALT1 karşı DNA probu basit PCR digoksijenin-dUTP birleşmeyle ile üretilmiştir. Daha sonra, bu TALT1 probu ve floresan işaretli telomer PNA probu aynı zamanda hibridize edildi. Daha sonra, digoksigenin kanonik immünofloresan yöntem ile tespit edilmiştir. Biz burada TALT1 TRT-1&#39;de telomer ile kolokalize temsili görüntüler sunmak </em&gt; kurtulanlar.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>PNA probe</td>
			<td>PANAGENE</td>
			<td>custom order</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-Digoxigenin-Fluorescein, Fab fragments</td>
			<td>Roche</td>
			<td>11207741910</td>
			<td>use 1:200 diluted in PBST</td>
		</tr>
		<tr>
			<td>Digoxigenin-dUTP</td>
			<td>Roche</td>
			<td>11573152910</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine serum albumin</td>
			<td>SIGMA-ALDRICH</td>
			<td>A-7906</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>SIGMA-ALDRICH</td>
			<td>P-6148</td>
			<td>prepare 4% paraformaldehyde by heating in DW with few drops of NaOH. add 0.1 volume of 10x PBS.</td>
		</tr>
		<tr>
			<td>Vectashield</td>
			<td>Vector Laboratories</td>
			<td>H-1200</td>
			<td></td>
		</tr>
		<tr>
			<td>Hybridizaiton solution</td>
			<td></td>
			<td></td>
			<td>3X SSC, 50% formamide, 10% (w/v) dextran sulfate, 50 ug/ml heparin, 100 ug/ml yeast tRNA , 100ug/ml sonicated salmon sperm DNA</td>
		</tr>
		<tr>
			<td>Hybridizaiton wash solution</td>
			<td></td>
			<td></td>
			<td>2X SSC, 50% formamide</td>
		</tr>
		<tr>
			<td>Formamide</td>
			<td>BIONEER</td>
			<td>C-9012</td>
			<td>toxic</td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td>Carlo Erba</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Acetone</td>
			<td>Carlo Erba</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Heparin</td>
			<td>SIGMA-ALDRICH</td>
			<td>H3393</td>
			<td>make 10 mg/ml for stock solution</td>
		</tr>
		<tr>
			<td>Dextran sulfate</td>
			<td>SIGMA-ALDRICH</td>
			<td>67578</td>
			<td></td>
		</tr>
		<tr>
			<td>10X PBS</td>
			<td></td>
			<td></td>
			<td>For 1 Liter DW : 80 g NaCl, 2.0 g KCl, 27 g Na2HPO4:7H2O, 2.4 g KH2PO</td>
		</tr>
		<tr>
			<td>PBST</td>
			<td></td>
			<td></td>
			<td>1X PBS, 0.1% tween-20</td>
		</tr>
		<tr>
			<td>Polysorbate 20</td>
			<td>SIGMA-ALDRICH</td>
			<td>P-2287</td>
			<td>Commercial name is Tween-20</td>
		</tr>
		<tr>
			<td>Poly-L-Lysine solution (0.1 % w/v)</td>
			<td>SIGMA-ALDRICH</td>
			<td>P-8920</td>
			<td>prepare fresh 0.01 % w/v solution before use</td>
		</tr>
		<tr>
			<td>M9</td>
			<td></td>
			<td></td>
			<td>3 g KH2PO4, 6 g Na2HPO4, 5 g NaCl, 1 ml 1 M MgSO4, H2O to 1 L</td>
		</tr>
		<tr>
			<td>Bleaching solution</td>
			<td></td>
			<td></td>
			<td>20% sodium hypochlorite, 0.5 M KOH</td>
		</tr>
		<tr>
			<td>Antibody buffer</td>
			<td></td>
			<td></td>
			<td>1X PBST, 1mM EDTA, 0.1% BSA, 0.05% Sodium azide (toxic)</td>
		</tr>
		<tr>
			<td>Blocking solution</td>
			<td></td>
			<td></td>
			<td>Antibody buffer with 5% bovine serum albumin (BSA)</td>
		</tr>
		<tr>
			<td>illustra Microspin G-50</td>
			<td>GE healthcare</td>
			<td>27-53310-01</td>
			<td></td>
		</tr>
		<tr>
			<td>20X SSC</td>
			<td></td>
			<td></td>
			<td>To make 1L, 175.3 g of NaCl, 88.2 g of sodium citrate, H2O to 1 L, adjust pH to 7.0</td>
		</tr>
		<tr>
			<td>2X SSCT</td>
			<td></td>
			<td></td>
			<td>2X SSC, 0.1 % tween-20</td>
		</tr>
		<tr>
			<td>10x digoxigenin-dUTP mix</td>
			<td></td>
			<td></td>
			<td>1 mM dATP, 1 mM dGTP, 1 mM dCTP, 0.65mM dTTP, 0.35mM DIG-11-dUTP</td>
		</tr>
		<tr>
			<td>PCR purification columns</td>
			<td>Cosmo genetech</td>
			<td>CMR0112</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass cleaner / ULTRA CLEAN</td>
			<td>Dukssan pure chemicals</td>
			<td>8AV721</td>
			<td></td>
		</tr>
		<tr>
			<td>Multi-well glass slide</td>
			<td>MP biomedicals</td>
			<td>96041205</td>
			<td></td>
		</tr>
		<tr>
			<td>Nematode growth media</td>
			<td></td>
			<td></td>
			<td>to make 1 L, 3 g of NaCl, 17 g of agar, 2.5 g of peptone, H2O to 974 mL. Autoclave and cool the flask. Add 1 mL of 1M CaCl2, 1 ml of 4 mg/mL cholesterol in ethanol, 1 ml of 1 M MgSO4, 25 mL of 1 M KPO4.</td>
		</tr>
		<tr>
			<td>Levamisole</td>
			<td>SIGMA-ALDRICH</td>
			<td>196142</td>
			<td></td>
		</tr>
		<tr>
			<td>Razor</td>
			<td>Feather</td>
			<td>blade No. 11</td>
			<td></td>
		</tr>
		<tr>
			<td>Rnase A</td>
			<td>Enzynomics</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>BSA</td>
			<td>SIGMA-ALDRICH</td>
			<td>A7906</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Equipments</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Confocal microsope</td>
			<td>Zeiss</td>
			<td>LSM 510</td>
			<td>EC Plan-Neofluar 100x was used as objective lens.</td>
		</tr>
		<tr>
			<td>Dry block / aluminum block</td>
			<td>Labtech</td>
			<td>LBH-T03</td>
			<td>Set temperature to 80&#8451;</td>
		</tr>
		<tr>
			<td>Humid chamber</td>
			<td></td>
			<td></td>
			<td>Plastic box filled with paper towel soaked in DW</td>
		</tr>
		<tr>
			<td>Image Analysis Software&#160;</td>
			<td>Dr. Peter Landsdorp</td>
			<td>TFL-telo</td>
			<td><a href="http://www.flintbox.com/public/project/502">http://www.flintbox.com/public/project/502</a></td>
		</tr>
	</tbody>
</table>

observation and quantification of telomere and repetitive sequences using fluorescence in situ hybridization (fish) with pna probes in caenorhabditis elegans

Telomere is a ribonucleoprotein structure that protects chromosomal ends from aberrant fusion and degradation. Telomere length is maintained by telomerase or an alternative pathway, known as alternative lengthening of telomeres (ALT)1. Recently, C. elegans has emerged as a multicellular model organism for the study of telomere and ALT2. Visualization of repetitive sequences in the genome is critical in understanding the biology of telomeres. While telomere length can be measured by telomere restriction fragment assay or quantitative PCR, these methods only provide the averaged telomere length. On the contrary, fluorescence in situ hybridization (FISH) can provide the information of the individual telomeres in cells. Here, we provide protocols and representative results of the method to determine telomere length of C. elegans by fluorescent in situ hybridization. This method provides a simple, but powerful, in situ procedure that does not cause noticeable damage to morphology. By using fluorescently labeled peptide nucleic acid (PNA) and digoxigenin-dUTP-labeled probe, we were able to visualize two different repetitive sequences: telomere repeats and template of ALT (TALT) in C. elegans embryos and gonads.

Daha önce ALT kurtulan içten &#39;ALT Şablon&#39; (tAlt) telomer bakım 2 dizileri lokalize çoğaltarak düşük frekansta, trt-1 (ok410), telomeraz eksikliği mutant ortaya çıkabileceği bildirildi. PNA probu kullanılarak, biz disseke gonadlar (Şekil 2A) &#39;de telomer görselleştirmek için başardık. Soluk telomer sinyali TRT-1 (ok410) ve ALT kurtulan hem de tespit edildi. Bulanık sinyal onlar otofloresans olmayabilir düşünd...

Watch this Scientific Journal Video about Observation and Quantification of Telomere and Repetitive Sequences Using Fluorescence In Situ Hybridization FISH with PNA Probes in Caenorhabditis elegans at

Observation and Quantification of Telomere and Repetitive Sequences Using Fluorescence In Situ Hybridization FISH with PNA Probes in Caenorhabditis elegans

We report a concise procedure of fluorescence in situ hybridization (FISH) in the gonad and embryos of Caenorhabditis elegans for observing and quantifying repetitive sequences. We successfully observed and quantified two different repetitive sequences, telomere repeats and template of alternative lengthening of telomeres (TALT).

Gözlem ve telomer Niceleme ve Tekrarlayan Diziler kullanma Floresan<em&gt; In Situ</emPNA Sondalar&gt; ile hibridizasyon (FISH)<em&gt; Caenorhabditis elegans</em

Telomere is a ribonucleoprotein structure that protects chromosomal ends from aberrant fusion and degradation. Telomere length is maintained by telomerase ...

observation-quantification-telomere-repetitive-sequences-using

Research

JoVE Journal

Biology

Observation and Quantification of Telomere and Repetitive Sequences Using Fluorescence In Situ Hybridization (FISH) with PNA Probes in Caenorhabditis elegans

10.3K Görüntüleme.  Seoul National University. We report a concise procedure of fluorescence in situ hybridization (FISH) in the gonad and embryos of Caenorhabditis elegans for observing and quantifying repetitive sequences. We successfully observed and quantified two different repetitive sequences, telomere repeats and template of alternative lengthening of telomeres (TALT). 

Video: Observation and Quantification of Telomere and Repetitive Sequences Using Fluorescence In Situ Hybridization FISH with PNA Probes in Caenorhabditis elegans

Fluorescence in situ hybridization (FISH) Protocol in Human Sperm

Aneuploidies are the most frequent chromosomal abnormalities in humans. Most of these abnormalities result from meiotic errors during the gametogenic process in the parents. In human males, these errors can lead to the production of spermatozoa with numerical chromosome abnormalities which represent an increased risk of transmitting these anomalies to the offspring.
For this reason, the technique of fluorescence in situ hybridization (FISH) on sperm nuclei has become a protocol widely incorporated in the context of clinical diagnosis. This practice provides an estimate of the frequencies of numerical chromosome abnormalities in the gametes of the patients that seek for genetic reproductive advice.
To date, the chromosomes most frequently included in sperm FISH analysis are chromosomes X, Y, 13, 18 and 21.
This video-article describes, step by step, how to process and fix a human semen sample, how to decondense and denature the sperm chromatin, how to proceed to obtain sperm FISH preparations, and how to visualize the results at the microscope. Special remarks of the most relevant steps are given to achieve the best results.

Fluorescence in situ hybridization FISH Protocol in Human Sperm

This video-article describes, step by step, how to process a semen sample to achieve good-quality fluorescence in situ hybridization on human spermatozoa. Preparations obtained can be used for aneuploidy screening in the context of clinical diagnosis.

Floresan In situ İnsan Sperm hibridizasyon (FISH) Protokolü

Aneuploidies are the most frequent chromosomal abnormalities in humans. Most of these abnormalities result from meiotic errors during the gametogenic ...

Biyoloji

Time-lapse Microscopy of Early Embryogenesis in Caenorhabditis elegans

Caenorhabditis elegans has often been used as a model system in studies of early developmental processes. The transparency of the 
embryos, the genetic resources, and the relative ease of transformation are qualities that make C. elegans an excellent model for early embryogenesis. Laser-based 
confocal microscopy and fluorescently labeled tags allow researchers to follow specific cellular structures and proteins in the developing embryo. For example, 
one can follow specific organelles, such as lysosomes or mitochondria, using fluorescently labeled dyes. These dyes can be delivered to the early embryo by means 
of microinjection into the adult gonad. Also, the localization of specific proteins can be followed using fluorescent protein tags. Examples are presented here 
demonstrating the use of a fluorescent lysosomal dye as well as fluorescently tagged histone and ubiquitin proteins. The labeled histone is used to visualize 
the DNA and thus identify the stage of the cell cycle. GFP-tagged ubiquitin reveals the dynamics of ubiquitinated vesicles in the early embryo. Observations 
of labeled lysosomes and GFP:: ubiquitin can be used to determine if there is colocalization between ubiquitinated vesicles and lysosomes. A technique for 
the microinjection of the lysosomal dye is presented. Techniques for generating transgenenic strains are presented elsewhere (1, 2). For imaging, embryos 
are cut out of adult hermaphrodite nematodes and mounted onto 2% agarose pads followed by time-lapse microscopy on a standard laser scanning confocal 
microscope or a spinning disk confocal microscope. This methodology provides for the high resolution visualization of early embryogenesis.

Time-lapse Microscopy of Early Embryogenesis in Caenorhabditis elegans

This article describes a technique for the visualization of the early events of embryogenesis in the nematode Caenorhabditis elegans.

Erken Embriyo Oluşumu Zaman atlamalı Mikroskopi Caenorhabditis elegans

Caenorhabditis elegans has often been used as a model system in studies of early developmental processes.  The transparency of the 
embryos, the genetic ...

Detection of Viral RNA by Fluorescence in situ Hybridization (FISH)

Viruses that infect cells elicit specific changes to normal cell functions which serve to divert energy and resources for viral replication. Many aspects of host cell function are commandeered by viruses, usually by the expression of viral gene products that recruit host cell proteins and machineries. Moreover, viruses engineer specific membrane organelles or tag on to mobile vesicles and motor proteins to target regions of the cell (during de novo infection, viruses co-opt molecular motor proteins to target the nucleus; later, during virus assembly, they will hijack cellular machineries that will help in the assembly of viruses). Less is understood on how viruses, in particular those with RNA genomes, coordinate the intracellular trafficking of both protein and RNA components and how they achieve assembly of infectious particles at specific loci in the cell. The study of RNA localization began in earlier work. Developing lower eukaryotic embryos and neuronal cells provided important biological information, and also underscored the importance of RNA localization in the programming of gene expression cascades. The study in other organisms and cell systems has yielded similar important information. Viruses are obligate parasites and must utilise their host cells to replicate. Thus, it is critical to understand how RNA viruses direct their RNA genomes from the nucleus, through the nuclear pore, through the cytoplasm and on to one of its final destinations, into progeny virus particles 1.
FISH serves as a useful tool to identify changes in steady-state localization of viral RNA. When combined with immunofluorescence (IF) analysis 22, FISH/IF co-analyses will provide information on the co-localization of proteins with the viral RNA3. This analysis therefore provides a good starting point to test for RNA-protein interactions by other biochemical or biophysical tests 4,5, since co-localization by itself is not enough evidence to be certain of an interaction. In studying viral RNA localization using a method like this, abundant information has been gained on both viral and cellular RNA trafficking events 6. For instance, HIV-1 produces RNA in the nucleus of infected cells but the RNA is only translated in the cytoplasm. When one key viral protein is missing (Rev) 7, FISH of the viral RNA has revealed that the block to viral replication is due to the retention of the HIV-1 genomic RNA in the nucleus 8. 
Here, we present the method for visual analysis of viral genomic RNA in situ. The method makes use of a labelled RNA probe. This probe is designed to be complementary to the viral genomic RNA. During the in vitro synthesis of the antisense RNA probe, the ribonucleotide that is modified with digoxigenin (DIG) is included in an in vitro transcription reaction. Once the probe has hybridized to the target mRNA in cells, subsequent antibody labelling steps (Figure 1) will reveal the localization of the mRNA as well as proteins of interest when performing FISH/IF.

Detection of Viral RNA by Fluorescence in situ Hybridization FISH

A fluorescence in situ hybridization (FISH) method was developed to visually detect viral genomic RNA using fluorescence microscopy. A probe is made with specificity to the viral RNA that can then be identified using a combination of hybridization and immunofluorescence techniques. This technique offers the advantage of identifying the localization of the viral RNA or DNA at steady-state, providing information on the control of intracellular virus trafficking events.

Floresans tarafından viral RNA tespit edilmesi<em&gt; In situ</em&gt; Hibridizasyon (FISH)

Viruses  that infect cells elicit specific changes to normal cell functions which serve  to divert energy and resources for viral replication. Many ...

Basic Caenorhabditis elegans Methods: Synchronization and Observation

Research into the molecular and developmental biology of the nematode Caenorhabditis elegans was begun in the early seventies by Sydney Brenner and it has since been used extensively as a model organism 1. C. elegans possesses key attributes such as simplicity, transparency and short life cycle that have made it a suitable experimental system for fundamental biological studies for many years 2. Discoveries in this nematode have broad implications because many cellular and molecular processes that control animal development are evolutionary conserved 3.
C. elegans life cycle goes through an embryonic stage and four larval stages before animals reach adulthood. Development can take 2 to 4 days depending on the temperature. In each of the stages several characteristic traits can be observed. The knowledge of its complete cell lineage 4,5 together with the deep annotation of its genome turn this nematode into a great model in fields as diverse as the neurobiology 6, aging 7,8, stem cell biology 9 and germ line biology 10. 
An additional feature that makes C. elegans an attractive model to work with is the possibility of obtaining populations of worms synchronized at a specific stage through a relatively easy protocol. The ease of maintaining and propagating this nematode added to the possibility of synchronization provide a powerful tool to obtain large amounts of worms, which can be used for a wide variety of small or high-throughput experiments such as RNAi screens, microarrays, massive sequencing, immunoblot or in situ hybridization, among others. 
Because of its transparency, C. elegans structures can be distinguished under the microscope using Differential Interference Contrast microscopy, also known as Nomarski microscopy. The use of a fluorescent DNA binder, DAPI (4',6-diamidino-2-phenylindole), for instance, can lead to the specific identification and localization of individual cells, as well as subcellular structures/defects associated to them.

Basic Caenorhabditis elegans Methods: Synchronization and Observation

The easiness of maintaining and propagating the nematode C. elegans make it a nice model organism to work with. The possibility of synchronizing worms allows the work with a significant amount of subjects at the same developmental stage, what facilitates the study of one particular process in many animals.

Temel<em&gt; Caenorhabditis elegans</em&gt; Yöntem: Senkronizasyon ve Gözlem

Research into the molecular and developmental biology of the nematode Caenorhabditis elegans was begun in the early seventies by Sydney Brenner and it has ...

Analysis of Single-cell Gene Transcription by RNA Fluorescent In Situ Hybridization (FISH)

Adhesion of Plasmodium falciparum infected erythrocytes (IE) to human endothelial receptors during malaria infections is mediated by expression of PfEMP1 protein variants encoded by the var genes.
The haploid P. falciparum genome harbors approximately 60 different var genes of which only one has been believed to be transcribed per cell at a time during the blood stage of the infection. How such mutually exclusive regulation of var gene transcription is achieved is unclear, as is the identification of individual var genes or sub-groups of var genes associated with different receptors and the consequence of differential binding on the clinical outcome of P. falciparum infections. Recently, the mutually exclusive transcription paradigm has been called into doubt by transcription assays based on individual P. falciparum transcript identification in single infected erythrocytic cells using RNA fluorescent in situ hybridization (FISH) analysis of var gene transcription by the parasite in individual nuclei of P. falciparum IE1.
Here, we present a detailed protocol for carrying out the RNA-FISH methodology for analysis of var gene transcription in single-nuclei of P. falciparum infected human erythrocytes. The method is based on the use of digoxigenin- and biotin- labeled antisense RNA probes using the TSA Plus Fluorescence Palette System2 (Perkin Elmer), microscopic analyses and freshly selected P. falciparum IE. The in situ hybridization method can be used to monitor transcription and regulation of a variety of genes expressed during the different stages of the P. falciparum life cycle and is adaptable to other malaria parasite species and other organisms and cell types.

Analysis of Single-cell Gene Transcription by RNA Fluorescent In Situ Hybridization FISH

Fluorescent in situ hybridization (FISH) to identify mRNA transcripts in individual cells allows analysis of polygenic activity such as the simultaneous transcription of more than one member of the var multigene family in Plasmodium falciparum infected erythrocytes 1. The technique is adaptable and can be used on different types of genes, cells and organisms.

RNA Floresan tarafından Tek hücreli Gen Transkripsiyon Analizi<em&gt; In Situ</em&gt; Hibridizasyon (FISH)

Adhesion of Plasmodium falciparum infected erythrocytes (IE) to human endothelial receptors during malaria infections is mediated by expression of PfEMP1 ...

Chromosome Replicating Timing Combined with Fluorescent In situ Hybridization

Mammalian DNA replication initiates at multiple sites along chromosomes at different times during S phase, following a temporal replication program. The specification of replication timing is thought to be a dynamic process regulated by tissue-specific and developmental cues that are responsive to epigenetic modifications. However, the mechanisms regulating where and when DNA replication initiates along chromosomes remains poorly understood. Homologous chromosomes usually replicate synchronously, however there are notable exceptions to this rule. For example, in female mammalian cells one of the two X chromosomes becomes late replicating through a process known as X inactivation1. Along with this delay in replication timing, estimated to be 2-3 hr, the majority of genes become transcriptionally silenced on one X chromosome. In addition, a discrete cis-acting locus, known as the X inactivation center, regulates this X inactivation process, including the induction of delayed replication timing on the entire inactive X chromosome. In addition, certain chromosome rearrangements found in cancer cells and in cells exposed to ionizing radiation display a significant delay in replication timing of &gt;3 hours that affects the entire chromosome2,3. Recent work from our lab indicates that disruption of discrete cis-acting autosomal loci result in an extremely late replicating phenotype that affects the entire chromosome4. Additional 'chromosome engineering' studies indicate that certain chromosome rearrangements affecting many different chromosomes result in this abnormal replication-timing phenotype, suggesting that all mammalian chromosomes contain discrete cis-acting loci that control proper replication timing of individual chromosomes5.
Here, we present a method for the quantitative analysis of chromosome replication timing combined with fluorescent in situ hybridization. This method allows for a direct comparison of replication timing between homologous chromosomes within the same cell, and was adapted from6. In addition, this method allows for the unambiguous identification of chromosomal rearrangements that correlate with changes in replication timing that affect the entire chromosome. This method has advantages over recently developed high throughput micro-array or sequencing protocols that cannot distinguish between homologous alleles present on rearranged and un-rearranged chromosomes. In addition, because the method described here evaluates single cells, it can detect changes in chromosome replication timing on chromosomal rearrangements that are present in only a fraction of the cells in a population.

Chromosome Replicating Timing Combined with Fluorescent In situ Hybridization

A quantitative method for the analysis of chromosome replication timing is described. The method utilizes BrdU incorporation in combination with fluorescent in situ hybridization (FISH) to assess replication timing of mammalian chromosomes. This technique allows for the direct comparison of rearranged and un-rearranged chromosomes within the same cell.

Floresan ile birlikte Kromozom çoğaltılıyor Zamanlama<em&gt; Yerinde</em&gt; Hibridizasyon

Mammalian DNA replication initiates at multiple sites along chromosomes at different times during S phase, following a temporal replication program. The ...

Visualization and Analysis of mRNA Molecules Using Fluorescence In Situ Hybridization in Saccharomyces cerevisiae

The Fluorescence in situ Hybridization (FISH) method allows one to detect nucleic acids in the native cellular environment. Here we provide a protocol for using FISH to quantify the number of mRNAs in single yeast cells. Cells can be grown in any condition of interest and then fixed and made permeable. Subsequently, multiple single-stranded deoxyoligonucleotides conjugated to fluorescent dyes are used to label and visualize mRNAs. Diffraction-limited fluorescence from single mRNA molecules is quantified using a spot-detection algorithm to identify and count the number of mRNAs per cell. While the more standard quantification methods of northern blots, RT-PCR and gene expression microarrays provide information on average mRNAs in the bulk population, FISH facilitates both the counting and localization of these mRNAs in single cells at single-molecule resolution.

Visualization and Analysis of mRNA Molecules Using Fluorescence In Situ Hybridization in Saccharomyces cerevisiae

This protocol describes an experimental procedure for performing Fluorescence in situ Hybridization (FISH) for counting mRNAs in single cells at single-molecule resolution.

Floresan kullanarak mRNA Moleküllerin Görselleştirme ve Analiz<em&gt; Yerinde</emIn&gt; Hibridizasyon<em&gt; Saccharomyces cerevisiae</em

The Fluorescence in situ Hybridization (FISH) method allows one to detect nucleic acids in the native cellular environment. Here we provide a protocol for ...

Prostaglandin Extraction and Analysis in Caenorhabditis elegans

Caenorhabditis elegans is emerging as a powerful animal model to study the biology of lipids1-9. Prostaglandins are an important class of eicosanoids, which are lipid signals derived from polyunsaturated fatty acids (PUFAs)10-14. These signalling molecules are difficult to study because of their low abundance and reactive nature. The characteristic feature of prostaglandins is a cyclopentane ring structure located within the fatty acid backbone. In mammals, prostaglandins can be formed through cyclooxygenase enzyme-dependent and -independent pathways10,15. C. elegans synthesizes a wide array of prostaglandins independent of cyclooxygenases6,16,17. A large class of F-series prostaglandins has been identified, but the study of eicosanoids is at an early stage with ample room for new discoveries. Here we describe a procedure for extracting and analyzing prostaglandins and other eicosanoids. Charged lipids are extracted from mass worm cultures using a liquid-liquid extraction technique and analyzed by liquid chromatography coupled to electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS). The inclusion of deuterated analogs of prostaglandins, such as PGF2 &alpha;-d4 as an internal standard is recommended for quantitative analysis. Multiple reaction monitoring or MRM can be used to quantify and compare specific prostaglandin types between wild-type and mutant animals. Collision-induced decomposition or MS/MS can be used to obtain information on important structural features. Liquid chromatography mass spectrometry (LC-MS) survey scans of a selected mass range, such as m/z 315-360 can be used to evaluate global changes in prostaglandin levels. We provide examples of all three analyses. These methods will provide researchers with a toolset for discovering novel eicosanoids and delineating their metabolic pathways.

Prostaglandin Extraction and Analysis in Caenorhabditis elegans

In this paper, we describe an optimized procedure for extracting and analyzing prostaglandins and other eicosanoids from C. elegans using LC-MS/MS.

Prostaglandin Ekstraksiyon ve Analiz<em&gt; Caenorhabditis elegans</em

Caenorhabditis elegans is emerging as a powerful animal model to study the biology of lipids1-9. Prostaglandins are an important class of eicosanoids, ...

Robust 3D DNA FISH Using Directly Labeled Probes

3D DNA FISH has become a major tool for analyzing three-dimensional organization of the nucleus, and several variations of the technique have been published. In this article we describe a protocol which has been optimized for robustness, reproducibility, and ease of use. Brightly fluorescent directly labeled probes are generated by nick-translation with amino-allyldUTP followed by chemical coupling of the dye. 3D DNA FISH is performed using a freeze-thaw step for cell permeabilization and a heating step for simultaneous denaturation of probe and nuclear DNA. The protocol is applicable to a range of cell types and a variety of probes (BACs, plasmids, fosmids, or Whole Chromosome Paints) and allows for high-throughput automated imaging. With this method we routinely investigate nuclear localization of up to three chromosomal regions.

We describe a robust and versatile protocol for analyzing nuclear architecture by 3D DNA FISH using directly labeled fluorescent probes.

Doğrudan Etiketli Problar kullanarak Sağlam 3D DNA BALIK

3D DNA FISH has become a major tool for  analyzing three-dimensional organization of the nucleus, and several variations  of the technique have been ...

Simple Method for Fluorescence DNA In Situ Hybridization to Squashed Chromosomes

DNA in situ hybridization (DNA ISH) is a commonly used method for mapping sequences to specific chromosome regions. This approach is particularly effective at mapping highly repetitive sequences to heterochromatic regions, where computational approaches face prohibitive challenges. Here we describe a streamlined protocol for DNA ISH that circumvents formamide washes that are standard steps in other DNA ISH protocols. Our protocol is optimized for hybridization with short single strand DNA probes that carry fluorescent dyes, which effectively mark repetitive DNA sequences within heterochromatic chromosomal regions across a number of different insect tissue types. However, applications may be extended to use with larger probes and visualization of single copy (non-repetitive) DNA sequences. We demonstrate this method by mapping several different repetitive sequences to squashed chromosomes from Drosophila melanogaster neural cells and Nasonia vitripennis spermatocytes. We show hybridization patterns for both small, commercially synthesized probes and for a larger probe for comparison. This procedure uses simple laboratory supplies and reagents, and is ideal for investigators who have little experience with performing DNA ISH.

Simple Method for Fluorescence DNA In Situ Hybridization to Squashed Chromosomes

Here, we present a simple method for performing fluorescence DNA in situ hybridization (DNA ISH) to visualize repetitive heterochromatic sequences on slide-mounted chromosomes. The method requires minimal reagents and it is versatile for use with short or long probes, different tissues, and detection with fluorescence or non-fluorescence-based signals.