Bu çalışma Charlotte, Kuzey Carolina Üniversitesi, öğretim mükemmellik için Wachovia vakıf fonu ve NIGMS (R15GM101571) hibe tarafından sağlanan fonların kısmen desteklenmektedir.

1. LSE Hazırlık <ol><li> Erkek kurbağalar (Xenopus laevis) yumurta toplama için iki kez enjekte edilir. İlk enjeksiyon (priming) kurbağa başına 100 U PMSG (Gebe Kısrak Serum Gonadotropin) &#39;dir. Indükleyici yumurtlama ve astarlanmış kurbağaları iki hafta için elverişlidir önce Kurbağalar en az iki gün astarlanmalıdır. Başbakan kurbağalar için, 3 ml şırınga ve 27 G iğne kullanılarak dorsal lenf torbalarıyla subkütan olarak PMSG enjekte. </li><li> Yumurtlama teşvik etmek için, 27 G iğne kullanılarak dorsal lenf torbalarıyla subkütan olarak astarlanmalı kurbağa başına 500 U hCG (insan koryonik gonadotropin) enjekte. 1x Marc modifiye Ringer solüsyonu (MMR) 2 litre içeren ayrı kovalarda enjekte kurbağa inkübe edin. Toplanmasından önce yumurtalarını bırakmak için kurbağa 14-20 saat bekleyin. </li><li> Kepçelerinden kurbağalar çıkarın ve yaklaşık 100 ml kalana kadar MMR çözümü kapalı dökün. 250 ml&#39;lik behere yumurta aktararak kovalar yumurta elde. </li><li> % 2 sistein, 100 ml (ayarlamak ekleyerek Dejelly yumurta10 M KOH ile 7.8 pH). Ters cam Pasteur pipeti (çapı 0.7 cm) yaklaşık her 30 sn yavaşça girdap yumurta. Durusu ve inkübasyon sırasında iki kez taze sistein ile değiştirin. Yumurta beherin altındaki bir daha yoğun bir tabaka oluşturur zaman dejellying işlem yaklaşık 5-15 dakika içinde tamamlandı. </li><li> Sistein solüsyonu atın ve 0.25x MMR ile yumurta üç kez yıkayın. Bir cam pipet ile çözelti içinde çalkalanarak yumurta. &quot;Bad&quot; yumurta basit görsel muayene ile tespit ve görünüş olarak &quot;kabarık&quot; beyaz vardır. &quot;Kötü&quot; yumurta girdapladıktan sonra çanağın merkezine birikir. Pasteur pipeti ile &quot;kötü&quot; yumurta çıkarın. </li><li> Yumurta Lizis Buffer (ELB) üç kez yumurta ile yıkayın. Pasteur pipeti tarafından herhangi bir ek &quot;kötü&quot; yumurta çıkarın. 14 ml Falcon tüp içine yumurta dökün. </li><li> Yumurta sıkıştırmak için bir sallanan kova rotor ile CL2 IEC klinik masa üstü santrifüj kullanılarak 188 xg (1.100 rpm) 55 sn için Falcon tüp Spin. Dan fazla tampon kaldırYumurta katmanın üstüne. Aprotinin / Löpeptin stokunun 0.5 ul ve sıkıştırılmış yumurta ml başına Sitokalazin B stokunun 0.5 ul ekleyin. </li><li> 4 az 15 dakika boyunca 16.500 xg&#39;de (10.000 rpm) yumurta santrifüjleyin ° C HB6 sallanan kepçe rotorlu Sorvall RC6 artı SuperSpeed ​​santrifüj kullanarak. Santrifüj sonra, yumurta tüp içinde üç katmandan fraksiyonlaşma: sırasıyla lipid, özü ve sarısı / üstten alta pigment. Bir 21 G iğne ile özü orta tabaka alt kısmında da delinme Falcon tüpün yan. Delme iğnesi plastik tarafından kesilebileceği gibi dikkatlice iğneyi çıkarın. Özü toplamak için ponksiyon içine 1 ml enjektöre bağlanmış taze 21 G iğne takın. Yavaşça lipid ve sarısı / pigment katmanları ile hava kabarcıkları ve kontaminasyon enjektöre özü aspire. </li><li> Soğutulmuş bir 1,5 ml mikrosantrifüj tüp içine özleri yerleştirin. Yumurta özü mililitre her biri için, içinde aşağıdaki kimyasal stok çözeltileri eklemekendikedir nihai konsantrasyon (parantez içinde gösterilmiştir): (1) 10 ul Sikloheksimit (100 ug / ml), (2) 1 ul Aprotinin / Löpeptin (10 ug / ml her biri), (3) 1 ul Sitokalazin B (5 ug / ml ), (4) 1 ul ditiyotretol (1 mM) ve (5) 0.33 ul Nokodazol (3 ug / ml). Yumurta hafifçe en az 10 kez ayıklamak Invert. Bu taze yapılmış ve 4 saat içinde kullanılmalıdır LSE vardır. LSE kalitesi 4 saat veya donma-çözülme sonrasında tehlikeye girer. </li></ol> 2. DNA Zararlı Yaklaşımlar ile Sperm Kromatin Tedavisi <ol><li> Daha önce 13 tarif edilen metoda göre normal sperm kromatin hazırlayın. </li><li> MMS ile tedavi edilen spermin kromatin hazırlayın <ol><li> 0.5 ml Tampon X&#39;de normal sperm kromatin süspanse </li><li> 100 mM nihai konsantrasyonu kromatin yeniden süspanse 500 ul MMS ~ 5.5 ul (stock 9.1 M) ekleyin. </li><li> Rotasyonu ile 30 dakika süreyle oda sıcaklığında mikrosantrifüj tüpü içinde sperm kromatin inkübe edin. </li><li> M Spinsallanan kova rotor ve boru adaptörü ile IEC CL2 klinik santrifüj kullanılarak oda sıcaklığında 10 dakika boyunca 686 x g (2100 rpm) icrocentrifuge tüpü. </li><li> Süpernatant atılır, Tampon X artı BSA (% 3), DTT (0.1 mM) ve aprotinin / Löpeptin (10 ug / ml her biri) 0.5 ml olarak pelet tekrar süspansiyon. </li><li> Adım (4) tekrarlayın ve (5) iki kez sperm kromatin yıkamak. </li><li> Hemasitometre ile sperm kromatin yoğunlaşması belirleyin ve 100.000 sperm / ul seyreltin. Daha fazla kullanım için -80 ° C derin dondurucuda 5 ul alikotları kaydedin. </li></ol></li><li> UV ile muamele edilen spermin kromatin hazırlayın <ol><li> Parafilm bir parça yüzeyine normal sperm kromatin istenilen miktarda (örneğin 10 ul) ekleyin. </li><li> UV çapraz bağlayıcı içine Parafilm yerleştirin. Denemenizi bağlı olarak istenilen enerji parametre, Set ve UV ışığı ile spermin kromatin zarar başlar. Örneğin, 1.000 J / m 2 &#39;ulaşmak için yaklaşık 21 saniye sürer. </li><li> UV irra sonradiation, UV ile muamele edilen sperm kromatin hemen yumurta özleri için ilave edilmelidir. </li></ol></li></ol> 3. DNA Hasarı-taklit Yapısının Hazırlanması (AT70) <ol><li> İki sentetik oligonükleotidler çözülür poli (dA) sırasıyla 70 (A70 olarak belirlenmiştir) ve poli-(dT) 70 (T70 olarak belirlenmiştir) 2 mg / ml &#39;lik bir konsantrasyon ile su içinde. </li><li> Bir 1.5 ml mikrosantrifüj tüpüne A70 100 ul ve 100 ul T70 ekleyin. Bir heatblock 95 5 dak ° C için karışık sentetik oligo kaynatıp. </li><li> Kuru banyo bloğu üzerinden (bu karışım AT içeren tüp ile) alınız ve bir laboratuar tezgah üzerinde oda sıcaklığına kadar soğumasını bekleyiniz. Bu sıcaklık ayarı 45-60 dakika sürer. </li><li> Soğutulan karışım, 2 ug / ul nihai konsantrasyon ile AT70 olup. Daha fazla kullanım için -20 ° C derin dondurucuda AT70 alikotları Mağaza 10 ul. </li></ol> 4. Hasarlı Sperm Kromatin veya DNA Hasarı ile LSE yılında DNA Hasarı Checkpoint Tetikleme-Taklit Yapısı <ol><li> Hasarlı sperm kromatin ile LSE DNA hasarı kontrol noktasında ikna <ol><li> 1,5 ml mikrosantrifüj tüp LSE 50 ul ekleyin ve Enerji Karışım 1 ul ve hasarlı sperm kromatin (son konsantrasyonu ~ 4.000 sperm / ul reaksiyon) 2 ul ile tamamlıyor. </li><li> 90 dakika süre ile oda sıcaklığında inkübe edin ve reaksiyon tüpü tüp her 10 dk hafifçe vurun. </li><li> 30 dakikalık inkübasyondan sonra nükleer boya çözeltisinin 1 ul ile desteklenmiş bir mikroskop lamı üzerine reaksiyon karışımı 1 ul dağıtın. Reaksiyon karışımı üzerinde bir kapak kayma yerleştirin ve floresan mikroskop aracılığıyla çekirdeklerin oluşumu için kontrol edin. Genellikle, yuvarlak nükleus DNA replikasyonu başlatmıştır gösteren, inkübasyon 30 dakika sonra oluşturacaktır. </li><li> Numune tamponu 90 ul reaksiyon karışımı içine 10 ul al. Numuneler anti-Chk1 P-S344 ya da anti-Chk1 antikorlar kullanılarak immünoblot vasıtasıyla analiz edilir. </li></ol></li><li> Ile LSE DNA hasar kontrol noktası indüklemekAT70 <ol><li> Enerji Karışım 1 ul ve Tautomycin stokunun 1.6 ul desteklenmiş 1.5 ml mikrosantrifüj tüp LSE 50 ul ekleyin. </li><li> Her reaksiyon için önceden hazırlanmış AT70 (2 ug / ul) ve su (negatif kontrol olarak) ve 1.25 ul ekle. 90 dakika için oda sıcaklığında reaksiyon inkübe edin. Reaksiyon tüpleri her 10 dk hafifçe vurun. </li><li> Numune tamponu 90 ul reaksiyon karışımı içine 10 ul ekle. Numuneler anti-Chk1 P-S344 ya da anti-Chk1 antikorlar kullanılarak immünoblot yoluyla incelenmiştir. </li></ol></li></ol> 5. Temsilcisi Sonuçlar Hasarlı sperm kromatin ya da DNA hasarı taklit eden yapısı Xenopus yumurta özü sisteminde ATR/Chk1-mediated DNA hasarı kontrol noktasında tetikleyebilir. MMS bir göstergesidir Ser344 az Chk1 fosforilasyon (Chk1 P-S344), uyardığı Şekil 2A gösterileri ATR kinaz aktivasyonu. Şekil 2B olduğunu AT70, bir DNA hasarı taklit eden stru gösterircture, ayrıca Chk1 fosforilasyon tetikler. Toplam Chk1 örnekler her iki örnekte de yükleme kontrol olarak kullanılmıştır. <img alt="Şekil 1" src="/files/ftp_upload/4449/4449fig1.jpg" /> Şekil 1. DNA hasarı kontrol noktasında sinyalizasyon bir diyagram. <img alt="Şekil 2," src="/files/ftp_upload/4449/4449fig2.jpg" /> Şekil 2. Chk1 fosforilasyon MMS veya Xenopus yumurta özler AT70 tedaviler ya tarafından indüklenir. (A) MMS-hasarlı sperm kromatin (MMS) veya normal sperm kromatin (Con) 90 dakika boyunca yumurta özleri içerisinde inkübe edilir. Chk1 Ser344 az fosforilasyon (Chk1 P-S344) ve yumurta özler toplam Chk1 immunoblotting yoluyla incelenmiştir. (B) ya da su AT70 (Con), sırasıyla, yumurta özleri içine ilave edilir. Örnekler ayrıca (A) &#39;de immünoblot vasıtasıyla analiz edilir.

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Çıkar çatışması ilan etti.

Xenopus yumurta özler kullanılarak DNA hasarı kontrol noktasında eğitim çeşitli avantajları vardır. Yumurta ekstrelerin hücre döngüsünün ara yüzeyinde eşzamanlı hücre içermeyen özler bir çok miktarda içerir. Yumurta ekstreler kolay ve ucuz hale getirilebilir. Bu DNA veya kromatin zarar ve yumurta özü bir hedef protein immunodepleting sonra DNA hasarı kontrol noktasında bir kusur ortaya nispeten kolaydır. Daha sonra, bir olası fonksiyon bozukluğu vahşi tip ya da mutant rekombinan pr...

<table border="1"><tbody><tr><td></td><td></td><td></td><td> Reaktifler </td></tr><tr><td> Anti-Chk1 P-S344 antikor </td><td> Hücre Sinyal </td><td> 2348L </td><td></td></tr><tr><td> Anti-Chk1 antikor </td><td> Santa Cruz </td><td> SC7898 </td><td></td></tr><tr><td> Aprotinin </td><td> MP Biomedicals </td><td> 0219115880 </td><td></td></tr><tr><td> Siklohekzimid </td><td> Sigma </td><td> C7698-5G </td><td></td></tr><tr><td> Sitokalazin B </td><td> EMD </td><td> 250233 </td><td></td></tr><tr><td> Ditiyotretol (DTT) </td><td> VWR </td><td> JTF780-2 </td><td></td></tr><tr><td> hCG </td><td> Sigma </td><td> CG10-10VL </td><td></td></tr><tr><td> L-Sistein </td><td> Sigma </td><td> C7352-1KG </td><td></td></tr><tr><td> Löpeptin </td><td> VWR </td><td>97063-922 </td><td></td></tr><tr><td> Metil metansülfonat (MMS) </td><td> Sigma </td><td> 129925-5G </td><td></td></tr><tr><td> Nokodazol </td><td> Sigma </td><td> M1404-2MG </td><td></td></tr><tr><td> PMSG </td><td> Calbiochem </td><td> 367222 </td><td></td></tr><tr><td> Numune tamponu </td><td> Sigma </td><td> S3401 </td><td></td></tr><tr><td> Tautomycin </td><td> Wako Kimyasallar Turkey </td><td> 209-12041 </td><td></td></tr><tr><td></td><td></td><td></td><td> Ekipman </td></tr><tr><td> Yumurtlama için Kepçe </td><td> Rubbermaid ticari ürünler </td><td> 6308 </td><td></td></tr><tr><td> Sallanan kepçe rotorlu CL2 IEC santrifüj </td><td> Thermo Scientific </td><td> 004260F </td><td></td></tr><tr><td> HB6 sallanan kova rotor </td><td> Thermo Scientific </td><td> 11860 </td><td></td></tr><tr><td> Sorvall RC6 artı SuperSpeed ​​santrifüj </td><td> Thermo Scientific </td><td> 46910 </td><td></td></tr><tr><td> UV çapraz bağlayıcı </td><td> UVP </td><td> 95-0174-01 </td><td></td></tr><tr><td></td><td></td><td></td><td> Çözümler </td></tr><tr><td> 1x MMR </td><td></td><td></td><td> 100 mM NaCl, 2 mM KCl, 0.5 mM MgSO4, 2.5 mM CaCl2, 5 mM HEPES, 10 M NaOH ile pH 7,8 &#39;e ayarlamak </td></tr><tr><td> Aprotinin / Löpeptin stok </td><td></td><td></td><td> 10 mg / ml her biri su içinde. -80 ° C&#39;de saklayın alikotları 20 ul </td></tr><tr><td> Tampon X </td><td></td><td></td><td> 0.2 M sukroz, 80 mM KCI, 15 mM NaCl, 5 mM MgCl2, 1 mM EDTA, 10 mM HEPES, HCl ile pH 7.5 &#39;e ayarlamak </td></tr><tr><td> Siklohekzimid stok </td><td></td><td></td><td> 1Su içinde 0 mg / ml. -20 ° C&#39;de saklayın 1 ml alikotları </td></tr><tr><td> Sitokalazin B stokları </td><td></td><td></td><td> DMSO içinde 5 mg / ml idi. -20 ° C&#39;de saklayın alikotları 20 ul </td></tr><tr><td> Ditiyotretol (DTT) stok </td><td></td><td></td><td> Su içinde 1 M. -20 ° C&#39;de saklayın 1 ml alikotları </td></tr><tr><td> ELB </td><td></td><td></td><td> 0.25 M sukroz, 1 mM DTT, 50 ug / ml sikloheksimid, 2.5 mM MgCl2, 50 mM KCI, 10 mM HEPES, pH7.7 </td></tr><tr><td> Nokodazol stok </td><td></td><td></td><td> DMSO içerisinde 10 mg / ml idi. -80 ° C&#39;de 5 ul alikot depolayın </td></tr><tr><td> Enerji Karışım </td><td></td><td></td><td> 375 mM kreatin fosfat, 50 mM ATP, 25 mM MgCl2. Alikotlar -80 ° C&#39;de saklanır </td></tr><tr><td> Nükleer boya çözeltisi </td><td></td><td></td><td> 0.4 mg / ml Hoechst 33258,% 25 gliserol(V / v) içinde 1 x PBS </td></tr><tr><td> Tautomycin stok </td><td></td><td></td><td> DMSO içinde 100 uM. -80 ° C&#39;de saklayın alikotları 10 ul </td></tr></tbody></table>

study of the dna damage checkpoint using xenopus egg extracts

Günlük olarak, hücreler, endojen ve çevresel hasarlar çeşitli tabi tutulur. Bu hakaretler mücadele etmek için, hücrelerin DNA hasarı ve DNA hasarı doğrudan hücresel yanıtları anlamda bir gözetim mekanizması olarak sinyalizasyon DNA hasarı kontrol noktasında gelişmiştir. DNA hasarı kontrol noktasında sinyal (Şekil 1) yer alan sensörler, transdüserler ve etkileyiciler adlandırılan proteinlerin birkaç grup vardır. Bu kompleks sinyal yolu olarak, ATR (ATM ve RAD3 ile ilgili) DNA hasarına ve çoğaltma strese cevap verebilecek kinazları önemli biridir. Aktif ATR gibi Chk1 olarak aşağı yüzeylerde (Checkpoint kinaz 1) fosforile edebilir. Sonuç olarak, fosforilasyona ve hücre döngüsünün durması, transkripsiyon aktivasyonu, DNA hasarının tamiri ve apoptosis ya da yaşlılık (Şekil 1) dahil olmak üzere DNA hasar kontrol noktası olarak çok sayıda alt-etkilere yol açar Chk1 aktive edildi. DNA hasar gördüğünde, unrep yılında DNA hasarı kontrol noktasında sonuçlar etkinleştirmek için başarısızyayınlanan hasar ve, sonradan, genomik instabilite. DNA hasarı kontrol noktasında çalışma hücrelerinin genomik bütünlüğünü korumak nasıl açıklamak ve kanser gibi insan hastalıkları, nasıl geliştiğini daha iyi anlaşılmasını sağlayacaktır. Xenopus laevis yumurta özleri DNA hasarı kontrol noktasında araştırma güçlü bir hücre serbest ekstresi model sistem olarak ortaya çıkmaktadır. Düşük hızda ekstresi (LSE) başlangıçta Masui grup 1 de tarif edilmiştir. DNA kez hücre döngüsü başına semiconservative moda çoğaltılır çekirdeklerin oluşumu LSE sonuçlarına demembranated spermin kromatin eklenmesi. ATR/Chk1-mediated noktası sinyal yolağı DNA hasarı veya çoğaltma stres 2 tarafından tetiklenir. DNA hasar yaklaşımlar ve DNA hasar taklit eden yapılar 3: iki yöntem, şu anda DNA hasar noktası indüklemek için kullanılır. DNA hasar ultraviyole (UV) ışın tedavisi, γ-ışınlama, metil metan indüklenebiliresulfonate (MMS), mitomisin C (MMC), 4-nitroquinoline-1-oksit (4-NQO), afidikodin ya da 3, 4. MMS DNA replikasyonu inhibe eder ve ATR/Chk1-mediated DNA hasar kontrol noktası 4-7 aktive edilen bir alkilleştirme ajanı. UV radyasyona da ATR/Chk1-dependent DNA hasar kontrol noktası 8 tetikler. DNA hasar taklit eden yapı AT70 iki oligonükleotidlerin poli (dA) 70 ve poli-(dT) 70 ve tavlanmış bir komplekstir. AT70 sistemi Bill Dunphy laboratuvarında geliştirilen ve yaygın olarak 9-12 ATR/Chk1 kapısı sinyalizasyon ikna etmek için kullanılır. Burada, (2) DNA hasarına taklit eden yapı hazırlamak için, iki farklı DNA zarar yaklaşımlar (PYS ve UV), (3) ile birlikte Xenopus sperm kromatin tedavi etmek için hücre içermeyen yumurta ekstreleri (LSE) maddesinin hazırlanması için protokolleri (1), tanımlamak AT70, ve (4) hasarlı sperm kromatin ya da bir DNA hasar taklit eden yapı ile LSE olarak ATR/Chk1-mediated DNA hasarını kontrol noktası tetikleyebilir.

Watch this Scientific Journal Video about Study of the DNA Damage Checkpoint using Xenopus Egg Extracts at JoVE.com

Study of the DNA Damage Checkpoint using Xenopus Egg Extracts

Xenopus yumurta özü DNA hasarı kontrol noktasında araştırmak için yararlı bir model sistemidir. Bu protokol, Xenopus yumurta özü ve DNA hasarına neden olan kontrol noktası reaktiflerin hazırlanması içindir. Bu teknikler DNA hasar kontrol noktası sinyalleşmenin çalışmada DNA hasarına yaklaşımların çeşitli uyarlanabilir.

Kullanılarak DNA hasar Checkpoint incelenmesi<em&gt; Xenopus</em&gt; Yumurta ayıklar

On a daily basis, cells are subjected to a variety of endogenous and environmental insults. To combat these insults, cells have evolved DNA damage ...

study-of-the-dna-damage-checkpoint-using-xenopus-egg-extracts

Research

JoVE Journal

Biology

Study of the DNA Damage Checkpoint using Xenopus Egg Extracts

16.6K Görüntüleme.  University of North Carolina at Charlotte. Xenopus yumurta özü DNA hasarı kontrol noktasında araştırmak için yararlı bir model sistemidir. Bu protokol, Xenopus yumurta özü ve DNA hasarına neden olan kontrol noktası reaktiflerin hazırlanması içindir. Bu teknikler DNA hasar kontrol noktası sinyalleşmenin çalışmada DNA hasarına yaklaşımların çeşitli uyarlanabilir.

Video: Study of the DNA Damage Checkpoint using Xenopus Egg Extracts

Testing Visual Sensitivity to the Speed and Direction of Motion in Lizards

Testing visual sensitivity in any species provides basic information regarding behaviour, evolution, and ecology. However, testing specific features of the visual system provide more empirical evidence for functional applications. Investigation into the sensory system provides information about the sensory capacity, learning and memory ability, and establishes known baseline behaviour in which to gauge deviations (Burghardt, 1977). However, unlike mammalian or avian systems, testing for learning and memory in a reptile species is difficult. Furthermore, using an operant paradigm as a psychophysical measure of sensory ability is likewise as difficult. Historically, reptilian species have responded poorly to conditioning trials because of issues related to motivation, physiology, metabolism, and basic biological characteristics. Here, I demonstrate an operant paradigm used a novel model lizard species, the Jacky dragon (Amphibolurus muricatus) and describe how to test peripheral sensitivity to salient speed and motion characteristics. This method uses an innovative approach to assessing learning and sensory capacity in lizards. I employ the use of random-dot kinematograms (RDKs) to measure sensitivity to speed, and manipulate the level of signal strength by changing the proportion of dots moving in a coherent direction. RDKs do not represent a biologically meaningful stimulus, engages the visual system, and is a classic psychophysical tool used to measure sensitivity in humans and other animals. Here, RDKs are displayed to lizards using three video playback systems. Lizards are to select the direction (left or right) in which they perceive dots to be moving. Selection of the appropriate direction is reinforced by biologically important prey stimuli, simulated by computer-animated invertebrates.

Testing visual sensitivity in lizards using an operant conditioning paradigm that employs video playback of random-dot kinematograms and computer-generated invertebrates.

Kertenkeleler Hareket Hız ve Yön Visual Hassasiyet Test

Testing visual sensitivity in any species provides basic information regarding behaviour, evolution, and ecology. However, testing specific features of ...

Biyoloji

Computer-Generated Animal Model Stimuli

Communication between animals is diverse and complex. Animals may communicate using auditory, seismic, chemosensory, electrical, or visual signals. In particular, understanding the constraints on visual signal design for communication has been of great interest. Traditional methods for investigating animal interactions have used basic observational techniques, staged encounters, or physical manipulation of morphology. Less intrusive methods have tried to simulate conspecifics using crude playback tools, such as mirrors, still images, or models. As technology has become more advanced, video playback has emerged as another tool in which to examine visual communication (Rosenthal, 2000). However, to move one step further, the application of computer-animation now allows researchers to specifically isolate critical components necessary to elicit social responses from conspecifics, and manipulate these features to control interactions. Here, I provide detail on how to create an animation using the Jacky dragon as a model, but this process may be adaptable for other species. In building the animation, I elected to use Lightwave  3D to alter object morphology, add texture, install bones, and provide comparable weight shading that prevents exaggerated movement. The animation is then matched to select motor patterns to replicate critical movement features. Finally, the sequence must rendered into an individual clip for presentation. Although there are other adaptable techniques, this particular method had been demonstrated to be effective in eliciting both conspicuous and social responses in staged interactions.

Computer-generated stimuli using the Jacky dragon as a model.

Bilgisayar oluşturuldu Hayvan Modeli Uyaranlar

Communication between animals is diverse and complex. Animals may communicate using auditory, seismic, chemosensory, electrical, or visual signals. In ...

Preparation and Fractionation of Xenopus laevis Egg Extracts

Crude and fractionated Xenopus egg extracts can be used to provide ingredients for reconstituting cellular processes for morphological and biochemical analysis. Egg lysis and differential centrifugation are used to prepare the crude extract which in turn in used to prepare fractionated extracts and light membrane preparations.

Crude and fractionated Xenopus egg extracts can be used to provide ingredients for reconstituting cellular processes for morphological and biochemical analysis. Egg lysis and differential centrifugation are used to prepare the crude extract which in turn in used to prepare fractionated extracts and light membrane preparations.

Xenopus laevis Yumurta hazırlanması ve Fraksiyonu ayıklar.

Crude and fractionated Xenopus egg extracts can be used to provide ingredients for reconstituting cellular processes for morphological and biochemical ...

In Vitro Nuclear Assembly Using Fractionated Xenopus Egg Extracts

Nuclear membrane assembly is an essential step in the cell division cycle; this process can be replicated in the test tube by combining Xenopus sperm chromatin, cytosol, and light membrane fractions. Complete nuclei are formed, including nuclear membranes with pore complexes, and these reconstituted nuclei are capable of normal nuclear processes.

Nuclear membrane assembly is an essential step in the cell division cycle; this process can be replicated in the test tube by combining Xenopus sperm chromatin, cytosol, and light membrane fractions. Complete nuclei are formed, including nuclear membranes with pore complexes, and these reconstituted nuclei are capable of normal nuclear processes.

Vitro Nükleer Meclisi fraksiyonlara Xenopus Yumurta ayıklar kullanma

Nuclear membrane assembly is an essential step in the cell division cycle; this process can be replicated in the test tube by combining Xenopus sperm ...

Fertilization of Xenopus oocytes using the Host Transfer Method

Studying the contribution of maternally inherited molecules to vertebrate early development is often hampered by the time and expense necessary to generate maternal-effect mutant animals. Additionally, many of the techniques to overexpress or inhibit gene function in organisms such as Xenopus and zebrafish fail to sufficiently target critical maternal signaling pathways, such as Wnt signaling. In Xenopus, manipulating gene function in cultured oocytes and subsequently fertilizing them can ameliorate these problems to some extent. Oocytes are manually defolliculated from donor ovary tissue, injected or treated in culture as desired, and then stimulated with progesterone to induce maturation. Next, the oocytes are introduced into the body cavity of an ovulating host female frog, whereupon they will be translocated through the host's oviduct and acquire modifications and jelly coats necessary for fertilization. The resulting embryos can then be raised to the desired stage and analyzed for the effects of any experimental perturbations. This host-transfer method has been highly effective in uncovering basic mechanisms of early development and allows a wide range of experimental possibilities not available in any other vertebrate model organism.

Fertilization of Xenopus oocytes using the Host Transfer Method

Procedure for fertilizing Xenopus oocytes by the host transfer method.

Gübrelenmesi Xenopus oositleri Host Transfer Yöntemi kullanılarak

Studying the contribution of maternally inherited molecules to vertebrate early development is often hampered by the time and expense necessary to ...

Two- and Three-Dimensional Live Cell Imaging of DNA Damage Response Proteins

Double-strand breaks (DSBs) are the most deleterious DNA lesions a cell can encounter. If left unrepaired, DSBs harbor great potential to generate mutations and chromosomal aberrations1. To prevent this trauma from catalyzing genomic instability, it is crucial for cells to detect DSBs, activate the DNA damage response (DDR), and repair the DNA. When stimulated, the DDR works to preserve genomic integrity by triggering cell cycle arrest to allow for repair to take place or force the cell to undergo apoptosis. The predominant mechanisms of DSB repair occur through nonhomologous end-joining (NHEJ) and homologous recombination repair (HRR) (reviewed in2). There are many proteins whose activities must be precisely orchestrated for the DDR to function properly. Herein, we describe a method for 2- and 3-dimensional (D) visualization of one of these proteins, 53BP1.
The p53-binding protein 1 (53BP1) localizes to areas of DSBs by binding to modified histones3,4, forming foci within 5-15 minutes5. The histone modifications and recruitment of 53BP1 and other DDR proteins to DSB sites are believed to facilitate the structural rearrangement of chromatin around areas of damage and contribute to DNA repair6. Beyond direct participation in repair, additional roles have been described for 53BP1 in the DDR, such as regulating an intra-S checkpoint, a G2/M checkpoint, and activating downstream DDR proteins7-9. Recently, it was discovered that 53BP1 does not form foci in response to DNA damage induced during mitosis, instead waiting for cells to enter G1 before localizing to the vicinity of DSBs6. DDR proteins such as 53BP1 have been found to associate with mitotic structures (such as kinetochores) during the progression through mitosis10.
In this protocol we describe the use of 2- and 3-D live cell imaging to visualize the formation of 53BP1 foci in response to the DNA damaging agent camptothecin (CPT), as well as 53BP1's behavior during mitosis. Camptothecin is a topoisomerase I inhibitor that primarily causes DSBs during DNA replication. To accomplish this, we used a previously described 53BP1-mCherry fluorescent fusion protein construct consisting of a 53BP1 protein domain able to bind DSBs11. In addition, we used a histone H2B-GFP fluorescent fusion protein construct able to monitor chromatin dynamics throughout the cell cycle but in particular during mitosis12. Live cell imaging in multiple dimensions is an excellent tool to deepen our understanding of the function of DDR proteins in eukaryotic cells.

This protocol describes a method for visualizing a DNA double-strand break signaling protein activated in response to DNA damage as well as its localization during mitosis.

DNA Hasarı Tepki Proteinler İki ve Üç Boyutlu Canlı Hücre Görüntüleme

Double-strand breaks (DSBs) are the most deleterious DNA lesions a cell can encounter. If left unrepaired, DSBs harbor great potential to generate ...

Production of Xenopus tropicalis Egg Extracts to Identify Microtubule-associated RNAs

Many organisms localize mRNAs to specific subcellular destinations to spatially and temporally control gene expression. Recent studies have demonstrated that the majority of the transcriptome is localized to a nonrandom position in cells and embryos. One approach to identify localized mRNAs is to biochemically purify a cellular structure of interest and to identify all associated transcripts. Using recently developed high-throughput sequencing technologies it is now straightforward to identify all RNAs associated with a subcellular structure. To facilitate transcript identification it is necessary to work with an organism with a fully sequenced genome. One attractive system for the biochemical purification of subcellular structures are egg extracts produced from the frog Xenopus laevis. However, X. laevis currently does not have a fully sequenced genome, which hampers transcript identification. In this article we describe a method to produce egg extracts from a related frog, X. tropicalis, that has a fully sequenced genome. We provide details for microtubule polymerization, purification and transcript isolation. While this article describes a specific method for identification of microtubule-associated transcripts, we believe that it will be easily applied to other subcellular structures and will provide a powerful method for identification of localized RNAs.

Production of Xenopus tropicalis Egg Extracts to Identify Microtubule-associated RNAs

We describe the collection of unfertilized Xenopus tropicalis eggs and production of a meiosis II-arrested egg extract. This egg extract can be used to purify microtubules and microtubule-associated RNAs.

Üretimi<em&gt; Xenopus tropicalis</em&gt; Yumurta Mikrotubul ilişkili RNA&#39;lar tanımak ayıklar

Many  organisms localize mRNAs to specific subcellular destinations to spatially and  temporally control gene expression. Recent studies have demonstrated ...

Reconstitution Of &#946;-catenin Degradation In Xenopus Egg Extract

Xenopus laevis egg extract is a well-characterized, robust system for studying the biochemistry of diverse cellular processes. Xenopus egg extract has been used to study protein turnover in many cellular contexts, including the cell cycle and signal transduction pathways1-3. Herein, a method is described for isolating Xenopus egg extract that has been optimized to promote the degradation of the critical Wnt pathway component, &beta;-catenin. Two different methods are described to assess &beta;-catenin protein degradation in Xenopus egg extract. One method is visually informative ([35S]-radiolabeled proteins), while the other is more readily scaled for high-throughput assays (firefly luciferase-tagged fusion proteins). The techniques described can be used to, but are not limited to, assess &beta;-catenin protein turnover and identify molecular components contributing to its turnover. Additionally, the ability to purify large volumes of homogenous Xenopus egg extract combined with the quantitative and facile readout of luciferase-tagged proteins allows this system to be easily adapted for high-throughput screening for modulators of &beta;-catenin degradation.

Reconstitution Of &#946;-catenin Degradation In Xenopus Egg Extract

A method is described for analyzing protein degradation using radiolabeled and luciferase-fusion proteins in Xenopus egg extract and its adaptation for high-throughput screening for small molecule modulators of protein degradation.

Β-katenin Bozulması Of Sulandırma<em&gt; Xenopus</em&gt; Yumurta Özü

Xenopus laevis egg extract is a well-characterized, robust system for studying the biochemistry of diverse cellular processes. Xenopus egg extract has ...

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

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

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

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

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

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

Amplification, Next-generation Sequencing, and Genomic DNA Mapping of Retroviral Integration Sites

Retroviruses exhibit signature integration preferences on both the local and global scales. Here, we present a detailed protocol for (1) generation of diverse libraries of retroviral integration sites using ligation-mediated PCR (LM-PCR) amplification and next-generation sequencing (NGS), (2) mapping the genomic location of each virus-host junction using BEDTools, and (3) analyzing the data for statistical relevance. Genomic DNA extracted from infected cells is fragmented by digestion with restriction enzymes or by sonication. After suitable DNA end-repair, double-stranded linkers are ligated onto the DNA ends, and semi-nested PCR is conducted using primers complementary to both the long terminal repeat (LTR) end of the virus and the ligated linker DNA. The PCR primers carry sequences required for DNA clustering during NGS, negating the requirement for separate adapter ligation. Quality control (QC) is conducted to assess DNA fragment size distribution and adapter DNA incorporation prior to NGS. Sequence output files are filtered for LTR-containing reads, and the sequences defining the LTR and the linker are cropped away. Trimmed host cell sequences are mapped to a reference genome using BLAT and are filtered for minimally 97% identity to a unique point in the reference genome. Unique integration sites are scrutinized for adjacent nucleotide (nt) sequence and distribution relative to various genomic features. Using this protocol, integration site libraries of high complexity can be constructed from genomic DNA in three days. The entire protocol that encompasses exogenous viral infection of susceptible tissue culture cells to integration site analysis can therefore be conducted in approximately one to two weeks. Recent applications of this technology pertain to longitudinal analysis of integration sites from HIV-infected patients.

We describe a protocol for amplifying retroviral integration sites from the genomic DNA of infected cells, sequencing the amplified virus-host junctions, and then mapping these sequences to a reference genome. We also describe techniques to quantify the distribution of integration sites relative to various genomic annotations using BEDTools.

Retroviral Entegrasyon Siteleri Amplifikasyon, Yeni Nesil Sıralama ve Genomik DNA Haritalama

Retroviruses exhibit signature integration preferences on both the local and global scales. Here, we present a detailed protocol for (1) generation of ...