Biz giriş ve Padilla Lab üyeleri yorumlarına için takdir ifade etmek istiyorum. Bu çalışmada Nematod suşları Araştırma Kaynakları için NIH Ulusal Merkezi (NCRR) tarafından finanse edilmektedir Caenorhabditis Genetik Merkezi tarafından sağlanmıştır. Biz konfokal mikroskopi ile teknik yardım için Dr Lon Turnbull kabul ve teşekkür ederim. Bu çalışma PAP Ulusal Bilim Vakfı (NSF-IOS, KARİYER) hibe tarafından desteklenmiştir

1. Örnek Hazırlanması <ol><li> Uygun transgenik C. üretin ya da elde elegans transgenik metodolojiler kullanarak faiz veya Caenorhabditis Genetik Stok Merkezi (CGC) veya iş arkadaşından süzün. Hücre bölünmesi için belirteç olarak kromozomlar ve sentrozomlarda görselleştirmek için 5; Bu durumda biz (pasta-1 :: GFP :: H2B pasta-1 :: TBG-1 :: GFP) suşu TH32 kullanıyorsunuz. </li><li> Üç yöntemden birini kullanarak senkronize bir nüfus oluşturun: 1) hipoklorit solüsyonu içinde gebe yetişkin parçalanır ve bir numaralı seribaşı tabağa gravid yetişkin almak ve onları 1-2 saat süreyle embriyolar bırakmanı kalan embriyoların 6, 2) kullanmak, veya 3) L4 almak Yeni bir numaralı seribaşı plaka karışık bir nüfus ve hareketten larva. </li><li> 20 nematod büyümek ° C L1 larvaları veya 24 saat sonrası L4 dökücü gelen, kuluçkalık 72 saat yaklaşık 96 saat sürecektir gravid genç yetişkinlik, için. Utero Embriyolar (2-20 hücre) imag için optimum olan büyük blastomer ve çekirdekler içerening alt hücresel ve alt nükleer değişiklikler, sırasıyla. Bu metodoloji erken embriyo bol sayıda içerebilir genç yetişkinden oluşan bir popülasyon oluşturmak için bir araç sağlar. </li></ol> 2. Slayt Hazırlama ve Oksijen yetersizliği Odası Kurma <ol><li> Yeterli büyüklükte bir kapak ile örtülen geniş bir Petri kabı veya bölme içinde nemli bir kağıt havlu koyarak hazırlanan slaytlar tutmak için nemli odasına olun. </li><li> Mikrodalga veya su banyosu yoluyla cam içinde karışımın ısıtılması ile deiyonize H 2 O erimiş% 2&#39;lik agaroz hazırlayın. </li><li> Temiz bir cam mikroskop lamı üzerine sıcak agaroz 1-3 damla yerleştirin. Hemen agaroz damla (lar) üstüne dik ters ikinci bir slayt yerleştirin çıkan pad ince ve hava kabarcıkları serbest olacak, biraz böylece basın. </li><li> Serin ve yavaş slaytlar birinde agaroz ped zedelemeden iki mikroskop slaytlar parçalara ayırmak için zaman tanıyınız. Bu agaroz ped etkileşimler için yeterli değildir ince olmasını sağlamak için önemlidirDaha sonra mikroskop odaklanma yeteneği ile yeniden. Örnek görselleştirmek becerisi de mikroskoplar arasında değişebilir kullanımda hedefi özel optik mesafe bağlıdır. </li><li> Küçük bir kare içine agaroz pedi şekillendirmek için temiz bir jilet kullanın. Kullanıma hazır olana kadar hazırlanan nemli odasında saklayın. </li><li> Dikkatle, aynı temiz jilet kullanarak, agaroz altlığın kenarına almak ve yuvarlak bir 25 mm mikro coverglass üzerine slayt aktarmanızı, ped altında hava kabarcıkları birikimi kaçınarak. Kullanmak üzere seçilen yuvarlak coverglass microincubator uygun şekilde uyuyor. </li><li> Bir anestetik olarak kullanım için agaroz ped üzerine anestetik (% 0.5 tricaine, M9 tamponu içinde% 0,05 Tetramisole), bir damla ekleme. Anestezik damla içine 5-10 solucanlar ve yeri seçin. Hareketi durdurma solucanlar için 1-3 dk bekleyin. </li><li> , Yetişkin rahim içinde embriyoların gözlemleyerek, yerine disseke embriyoların gerekçesi embriyo desicca potansiyelini en aza indirmek içintion ve embriyo üzerinde azot gazı akışına bağlı hareket. </li><li> Hemen gaz odasına akardı olarak dehidratasyon solucanları önlemek için solucanlar üstüne Halokarbon bir damla yağ ekleyin. Halokarbon yağ viskoz olduğu tek solucanlar üzerine yağ ve damla toplamak için almak bir solucanın bir pipet ucu veya ipucu kullanabilirsiniz. </li><li> Solucanlar içeren dairesel coverglass hazırlandıktan sonra, Leiden iki yarısı microincubator ayrılır perfüzyon kapalı ve coverglass sonra dikkatlice alt halkaya dik yerleştirilir. Odanın iki tarafı da birbirine sıkıca kapatılır. </li><li> Oda sonra esnek plastik borular vasıtasıyla nitrojen gazı tanka bağlanmış ve mikroskop aşamada (Şekil 1C) üzerinde uygun noktaya yerleştirilir. Şu anda tüp ve microincubator dikkatle tüp ve odanın yan tarafındaki bağlantı fişleri güvenli bağlanma sağlayarak herhangi bir sızıntı olup olmadığını kontrol edilmelidir. Bir al edebilirsinizbu yüzden hafif bir sızıntı varsa oluşturacak kabarcıklar, aramak için tüp üzerinde sabun ve su küçük bir miktar yayılır. </li></ol> 3. Oksijen yetersizliği yılında Mikroskopi <ol><li> Alt büyütmede hayvanlar bulun, sonra, görüntü doku ya da bu gibi yetişkin embriyonik blastomerlerin veya oosit olarak ilgi hücreleri için uygun daha yüksek bir büyütmede (bu uygulama için 64x) getirin. GFP sinyali görüntülemek ve faiz hücre (ler) GFP füzyon proteini bulmak için 488 nm lazer açın. </li><li> Hücre döngüsü tutuklama belirli aşamasında tutuklama görselleştirmek için (örn. geç profaz) hücre bölünmesi önceki bir aşamada (örneğin geç interfaz ya da erken profaz) bir blastomer tanımlamak. Böyle sentriol konumu, kromozom kondensasyonu başlaması, kromozom konumu ve nükleer zarfın varlığı veya yokluğu gibi Hücresel belirteçler hücre döngüsü aşamada (Şekil 2A) belirlemenize yardımcı olacaktır. </li><li> Kamara, nitrojen gazı (% 99.999 ile perfüze edilmiştirN 2; &lt;2 ppm O 2). Biz 6 psi arasında bir basınç kullanımı bu durumda, ancak basınç ayarlanmış ve her bir microincubator boyutu ve kullanım boru çapına bağlı olarak, optimize edilmesi gerekebilir. Azot gerekli odak düzlemi ayarlayarak, kamara doldurur gibi blastomer (ler) izlemek için devam edin. </li><li> Bu noktada, time-lapse görüntüleme başladı. Görüntüleme iç nükleer membran kromozomların bu durumda profaz tutuklama ve yerleştirme yılında, faiz olgusu yakalamak için gerektiği kadar için yapılır. Görüntü 30 dakika daha uzun süreyle her 10 saniyede bir alınır. Görüntü yakalama ve bu kazanç ve lazer güç gibi ayarları Frekanslar gerektiğinde ayarlanmalıdır. </li><li> Embriyoların anoksik ortamda yanıt hangi zaman anoksi maruz kalan hücre döngüsü evresine göre değişebilir. Genellikle anoksi indüklenen profaz tutuklama azot gazı akışı başladıktan 20-30 dakika içinde oluşur. Biz anoksi indüklenen hücre döngüsü tutuklama occu gözlemledik&lt;20 dk içinde rring. Resimler bu süre esnasında elde edilebilir, ya da belirli görüntü daha sonra bir zaman ayarlı seri izole edilebilir. Ek bir seçenek embriyo alt hücresel yapılar görselleştirmek için video mikroskopi kullanmaktır. </li><li> Anoksi bağlı tutuklandı devleti kurtarma belgelemek için, gaz kapalı ve odasına bir time-lapse serisi kaydederken difüzyon ile normoksi dönmek için izin verilir. Kromozomların hücre siklus progresyonu ve undocking Sürmesine genellikle azot gazı kapatarak 5-20 dakika içinde gerçekleşir. </li><li> Ayrı deneylerde bir anoksi göstergesi resazurin, flow-through microchamber yerleştirildi unutmayın; bunun azot gazı akışı başladıktan sonra odasına yaklaşık 19 dakika ortalama anoksik hale belirlenmiştir. </li><li> Görüntüler ve videolar Imaris, Görüntü J veya Photoshop kullanarak işlenir ve görüntüleme için QuickTime alınır. </li></ol>

<ol>
	<li>Powell-Coffman, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hypoxia+signaling+and+resistance+in+C.+elegans.">Hypoxia signaling and resistance in C. elegans.</a> Trends Endocrinol. Metab. 21 (10), 435-440 (2010).</li><li>Padilla, P. A., Ladage, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Suspended+animation,+diapause+and+quiescence:+Arresting+the+cell+cycle+in+C.+elegans.">Suspended animation, diapause and quiescence: Arresting the cell cycle in C. elegans.</a> Cell Cycle. 11, (2012).</li><li>Hu, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dauer.">Dauer.</a> WormBook. , 1-19 (2007).</li><li>Zhou, K. I., Pincus, Z., Slack, F. 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B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The Nematode Caenorhabditis elegans. , 587-606 (1988).</li><li>Padilla, P. A., Nystul, T. G., Zager, R. A., Johnson, A. C., Roth, M. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dephosphorylation+of+Cell+Cycle-regulated+Proteins+Correlates+with+Anoxia-induced+Suspended+Animation+in+Caenorhabditis+elegans.">Dephosphorylation of Cell Cycle-regulated Proteins Correlates with Anoxia-induced Suspended Animation in Caenorhabditis elegans.</a> Mol. Biol Cell. 13, 1473-1483 (2002).</li><li>Hajeri, V. A., Trejo, J., Padilla, P. 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B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Suspended+animation+in+C.+elegans+requires+the+spindle+checkpoint.">Suspended animation in C. elegans requires the spindle checkpoint.</a> Science. 302, 1038-1041 (2003).</li><li>Margalit, A., Vlcek, S., Gruenbaum, Y., Foisner, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Breaking+and+making+of+the+nuclear+envelope.">Breaking and making of the nuclear envelope.</a> J. Cell Biochem. 95, 454-465 (2005).</li><li>Maddox, A. S., Maddox, P. 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Çıkar çatışması ilan etti.

Oksijen Yoksunluğu ve Asma Animasyon Ciddi oksijen yoksunluğu maruz bazı organizmalar için ölümcül olabilir rağmen, bazı organizmalar anoksi maruz hayatta edebiliyoruz. C. halinde elegans, anoksi maruziyet sağkalım gelişim safhasına ve anoksi yanıtın bağlıdır girişi gözlemlenebilir biyolojik süreçlerin bir dizi tutuklandı edildiği askıya animasyon bir tersinir durum içine. Oksijen ortamında 7,9 tekrar geri kadar Hücre bölünmesi, gelişm...

<table border="1"><tbody><tr><td> Reaktifler / Ekipman </td><td></td><td></td><td> Kompozisyon </td></tr><tr><td> Hipoklorit solüsyonu </td><td></td><td></td><td> 0.7 g KOH, 12 ml% 5 NaOCİ, GKD 2 0 ile 50 ml&#39;ye getirmek </td></tr><tr><td> M9 tampon </td><td></td><td></td><td> 3 g KH 2 PO 4, 11 gr Na 2 HPO 4, 5 g NaCl, 1 L GKD 2 O başına 1 ml (1 M) MgSO4 </td></tr><tr><td> Cam mikroskop slaytlar </td><td> Fisher Scientific </td><td> 12-550-343 </td><td> 3 &quot;x1&quot; x1.0 mm </td></tr><tr><td> Yuvarlak mikro coverglass </td><td> Elektron Mikroskopi Bilimleri </td><td> 72223-01 </td><td> 25 mm Çap </td></tr><tr><td> Halokarbon yağı 700 </td><td> Sigma </td><td> H8898-100ml </td><td></td></tr><tr><td> Anestetik </td><td></td><td></td><td> % 0.5 gastrikaine,% 0.05 Tetramisole </td></tr><tr><td> Leiden Kapalı Perfüzyon Microincubator </td><td> Harvard Apparatus </td><td> 650041 </td><td></td></tr><tr><td> UHP Azot </td><td> Calgaz (Air Liquide) </td><td></td><td> &gt; 99,9990% N 2 &lt;2 ppm O 2 </td></tr><tr><td> Plastik boru </td><td> VWR </td><td> 89068-468 </td><td> 0.062 &quot;ID x 0.125&quot; OD </td></tr><tr><td> Disk Konfokal Mikroskop Spinning </td><td> McBain Sistemleri </td><td></td><td> Zeiss optik mikroskop, Epifloresans aydınlatma sistemi, CSU-10 Yokogawa konfokal tarayıcı, Hamamatsu elektron çoğaltıcı CCD kamera ters. </td></tr></tbody></table>

use of time lapse microscopy to visualize anoxia-induced suspended animation in c. elegans embryos

Caenorhabdits elegans yetişkin ve embriyo aşamalarında şeffaflık yanı sıra füzyon proteinleri 1-4 sayısız ifade genetik mutantlar ve transgenik suşların durumu kolaylaştırılır strese dayanıklılığı çalışmalarında yaygın olarak kullanılmaktadır. Ayrıca, hücre bölünmesi gibi dinamik süreçleri floresan etiketli muhabiri proteinleri kullanılarak izlenebilir. Mitoz çalışma dokunulmamış organizmalar dahil olmak üzere çok çeşitli sistemler de hızlandırılmış deneylerinin kullanılmasıyla kolaylaştırılabilir ve böylece ilk C. elegans embriyo de bu çalışma için uygundur. Yüksek güçlü bir mikroskop kurulum ile basit bir gaz akışı kullanarak stres mümkündür; Sunan burada anoksik yanıt (&lt;2 ppm O 2% 99.999 N 2) alt hücresel yapıların in vivo görüntüleme hangi bir tekniktir. Bir microincubation odasından geçen azot gazı akışı ve dönen bir disk konfokal mikroskop ile birlikte kullanılırhayvanlarda in vivo yansıması hangi kontrollü bir ortam oluşturmak için. Kullanma GFP etiketli gama tübülin ve histon, dinamikleri ve hücre bölünmesi tutuklama sırasında ve bir oksijen-yoksun ortama maruz kaldıktan sonra, önce izlenebilir. Bu tekniğin sonuçları oksijen yoksunluğu maruz embriyoların blastomer içinde yüksek çözünürlüklü, ayrıntılı video ve hücresel yapıların görüntülerdir.

Ciddi oksijen yoksunluğu (anoksi) maruz C. elegans embriyo gelişimi ve hücre bölünmesi 7 dahil biyolojik süreçleri tutuklayarak hayatta edebiliyoruz. Hücre bölünmesi anoksi indüklenen tutuklama hayvanlarda in vivo yansıması hangi kontrollü bir ortam yaratmak yoluyla azot gazı akışı ve dönen bir disk konfokal mikroskop ile birlikte bir microincubation odası kullanarak, bir alt-hücresel düzeyde, izlenebilir (Şekil 1). Ilgi Hücresel yapıları çeşitli ...

Watch this Scientific Journal Video about Use of Time Lapse Microscopy to Visualize Anoxia-induced Suspended Animation in C. elegans Embryos at JoVE.com

Use of Time Lapse Microscopy to Visualize Anoxia-induced Suspended Animation in C. elegans Embryos

Bir burada tarif edilmekte<em&gt; In vivo</emBir dönen diske konfokal mikroskop ile birlikte microincubation odasından geçen bir gaz akımı kullanılarak anoksi maruz kalan hayvanlarda resim alt hücresel yapılara&gt; tekniktir. Bu yöntem basit ve deneysel parametreleri ve model sistemleri çeşitli uyacak kadar esnek.

In Oksijen yetersizliği kaynaklı Asma Animasyon Visualize&#39;a Time Lapse Mikroskopi kullanın<em&gt; C. elegans</em&gt; Embriyolar

Caenorhabdits elegans has been used extensively in the study of stress resistance, which is facilitated by the transparency of the adult and embryo stages ...

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JoVE Journal

Biology

Use of Time Lapse Microscopy to Visualize Anoxia-induced Suspended Animation in C. elegans Embryos

11.5K Görüntüleme.  University of North Texas. Bir burada tarif edilmekte In vivo tekniktir. Bu yöntem basit ve deneysel parametreleri ve model sistemleri çeşitli uyacak kadar esnek.

Video: Use of Time Lapse Microscopy to Visualize Anoxia-induced Suspended Animation in C. elegans Embryos

Monitoring Actin Disassembly with Time-lapse Microscopy

Zaman atlamalı Mikroskopi Sökme Aktin İzlenmesi

Biyoloji

Two-photon axotomy and time-lapse confocal imaging in live zebrafish embryos

Zebrafish have long been utilized to study the cellular and molecular mechanisms of development by time-lapse imaging of the living transparent embryo. Here we describe a method to mount zebrafish embryos for long-term imaging and demonstrate how to automate the capture of time-lapse images using a confocal microscope. We also describe a method to create controlled, precise damage to individual branches of peripheral sensory axons in zebrafish using the focused power of a femtosecond laser mounted on a two-photon microscope. The parameters for successful two-photon axotomy must be optimized for each microscope. We will demonstrate two-photon axotomy on both a custom built two-photon microscope and a Zeiss 510 confocal/two-photon to provide two examples.

Zebrafish trigeminal sensory neurons can be visualized in a transgenic line expressing GFP driven by a sensory neuron specific promoter 1. We have adapted this zebrafish trigeminal model to directly observe sensory axon regeneration in living zebrafish embryos. Embryos are anesthetized with tricaine and positioned within a drop of agarose as it solidifies. Immobilized embryos are sealed within an imaging chamber filled with phenylthiourea (PTU) Ringers. We have found that embryos can be continuously imaged in these chambers for 12-48 hours. A single confocal image is then captured to determine the desired site of axotomy. The region of interest is located on the two-photon microscope by imaging the sensory axons under low, non-damaging power. After zooming in on the desired site of axotomy, the power is increased and a single scan of that defined region is sufficient to sever the axon. Multiple location time-lapse imaging is then set up on a confocal microscope to directly observe axonal recovery from injury.

Here we describe a method for mounting zebrafish embryos for long-term imaging, two-photon imaging and tissue-damage techniques, and time-lapse confocal imaging.

İki foton axotomy ve zaman atlamalı konfokal görüntüleme canlı zebrafish embriyolar

Zebrafish have long been utilized to study the cellular and molecular mechanisms of development by time-lapse imaging of the living transparent embryo.  ...

Time-lapse Imaging of Mitosis After siRNA Transfection

Changes in cellular organization and chromosome dynamics that occur during mitosis are tightly coordinated to ensure accurate inheritance of genomic and cellular content. Hallmark events of mitosis, such as chromosome movement, can be readily tracked on an individual cell basis using time-lapse fluorescence microscopy of mammalian cell lines expressing specific GFP-tagged proteins. In combination with RNAi-based depletion, this can be a powerful method for pinpointing the stage(s) of mitosis where defects occur after levels of a particular protein have been lowered. In this protocol, we present a basic method for assessing the effect of depleting a potential mitotic regulatory protein on the timing of mitosis. Cells are transfected with siRNA, placed in a stage-top incubation chamber, and imaged using an automated fluorescence microscope. We describe how to use software to set up a time-lapse experiment, how to process the image sequences to make either still-image montages or movies, and how to quantify and analyze the timing of mitotic stages using a cell-line expressing mCherry-tagged histone H2B. Finally, we discuss important considerations for designing a time-lapse experiment. This strategy is complementary to other approaches and offers the advantages of 1) sensitivity to changes in kinetics that might not be observed when looking at cells as a population and 2) analysis of mitosis without the need to synchronize the cell cycle using drug treatments. The visual information from such imaging experiments not only allows the sub-stages of mitosis to be assessed, but can also provide unexpected insight that would not be apparent from cell cycle analysis by FACS.

Here we describe a basic protocol to image and quantify the mitotic timing of live mammalian tissue culture cells after siRNA transfection.

Zaman atlamalı Görüntüleme siRNA Transfeksiyon sonra Mitoz

Changes in cellular organization and chromosome dynamics that occur during mitosis are tightly coordinated to ensure accurate inheritance of genomic and ...

Imaging C. elegans Embryos using an Epifluorescent Microscope and Open Source Software

Cellular processes, such as chromosome assembly, segregation and cytokinesis,are inherently dynamic. Time-lapse imaging of living cells, using fluorescent-labeled reporter proteins or differential interference contrast (DIC) microscopy, allows for the examination of the temporal progression of these dynamic events which is otherwise inferred from analysis of fixed samples1,2. Moreover, the study of the developmental regulations of cellular processes necessitates conducting time-lapse experiments on an intact organism during development. The Caenorhabiditis elegans embryo is light-transparent and has a rapid, invariant developmental program with a known cell lineage3, thus providing an ideal experiment model for studying questions in cell biology4,5and development6-9. C. elegans is amendable to genetic manipulation by forward genetics (based on random mutagenesis10,11) and reverse genetics to target specific genes (based on RNAi-mediated interference and targeted mutagenesis12-15). In addition, transgenic animals can be readily created to express fluorescently tagged proteins or reporters16,17. These traits combine to make it easy to identify the genetic pathways regulating fundamental cellular and developmental processes in vivo18-21. In this protocol we present methods for live imaging of C. elegans embryos using DIC optics or GFP fluorescence on a compound epifluorescent microscope. We demonstrate the ease with which readily available microscopes, typically used for fixed sample imaging, can also be applied for time-lapse analysis using open-source software to automate the imaging process.

Imaging C. elegans Embryos using an Epifluorescent Microscope and Open Source Software

The C. elegans embryo is a powerful system for studying cell biology and development. We present a protocol for live imaging of C. elegans embryos utilizing DIC optics or fluorescence using readily available epifluorescent microscopes and open-source software.

Görüntüleme C. elegans Embriyolar Epifluorescent Mikroskop ve Açık Kaynak Yazılım

Cellular processes, such as chromosome assembly, segregation and cytokinesis,are inherently dynamic. Time-lapse imaging of living cells, using ...

Determining Genetic Expression Profiles in C. elegans Using Microarray and Real-time PCR

Synapses are composed of a presynaptic active zone in the signaling cell and a postsynaptic terminal in the target cell. In the case of chemical synapses, messages are carried by neurotransmitters released from presynaptic terminals and received by receptors on postsynaptic cells. Our previous research in Caenorhabditis elegans has shown that VSM-1 negatively regulates exocytosis. Additionally, analysis of synapses in vsm-1 mutants showed that animals lacking a fully functional VSM-1 have increased synaptic connectivity. Based on these preliminary findings, we hypothesized that C. elegans VSM-1 may play a crucial role in synaptogenesis. To test this hypothesis, double-labeled microarray analysis was performed, and gene expression profiles were determined. First, total RNA was isolated, reversely transcribed to cDNA, and hybridized to the DNA microarrays. Then, in-silico analysis of fluorescent probe hybridization revealed significant induction of many genes coding for members of the major sperm protein family (MSP) in mutants with enhanced synaptogenesis. MSPs are the major component of sperm in C. elegans and appear to signal nematode oocyte maturation and ovulation . In fruit flies, Chai and colleagues 1 demonstrated that MSP-like molecules regulate presynaptic bouton number and size at the neuromuscular junction. Moreover, analysis performed by Tsuda and coworkers 2 suggested that MSPs may act as ligands for Eph receptors and trigger receptor tyrosine kinase signaling cascades. Lastly, real time PCR analysis corroborated that the gene coding for MSP-32 is induced in vsm-1(ok1468) mutants. Taken together, research performed by our laboratory has shown that vsm-1 mutants have a significant increase in synaptic density, which could be mediated by MSP-32 signaling.

Determining Genetic Expression Profiles in C. elegans Using Microarray and Real-time PCR

Microarray analysis was conducted to determine genetic expression profiles in C. elegans, and real-time PCR was used to validate and quantify microarray data.

Genetik İfade Profilleri Belirlenmesi C. elegans Mikroarray ve Real-time PCR

Synapses are composed of a presynaptic active zone in the signaling cell and a postsynaptic terminal in the target cell. In the case of chemical synapses, ...

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 ...

RNAi Screening to Identify Postembryonic Phenotypes in C. elegans

C. elegans has proven to be a valuable model system for the discovery and functional characterization of many genes and gene pathways1. More sophisticated tools and resources for studies in this system are facilitating continued discovery of genes with more subtle phenotypes or roles. 
Here we present a generalized protocol we adapted for identifying C. elegans genes with postembryonic phenotypes of interest using RNAi2. This procedure is easily modified to assay the phenotype of choice, whether by light or fluorescence optics on a dissecting or compound microscope. This screening protocol capitalizes on the physical assets of the organism and molecular tools the C. elegans research community has produced. As an example, we demonstrate the use of an integrated transgene that expresses a fluorescent product in an RNAi screen to identify genes required for the normal localization of this product in late stage larvae and adults. First, we used a commercially available genomic RNAi library with full-length cDNA inserts. This library facilitates the rapid identification of multiple candidates by RNAi reduction of the candidate gene product. Second, we generated an integrated transgene that expresses our fluorecently tagged protein of interest in an RNAi-sensitive background. Third, by exposing hatched animals to RNAi, this screen permits identification of gene products that have a vital embryonic role that would otherwise mask a post-embryonic role in regulating the protein of interest. Lastly, this screen uses a compound microscope equipped for single cell resolution.

RNAi Screening to Identify Postembryonic Phenotypes in C. elegans

We describe a sensitized method to identify postembryonic regulators of protein expression and localization in C. elegans using an RNAi-based genomic screen and an integrated transgene that expresses a functional, fluorescently tagged protein.

Içinde Postembryonic Fenotiplerin Belirleme RNAi Tarama C. elegans</em

C. elegans has proven to be a valuable model system for the discovery and functional characterization of many genes and gene pathways1. More sophisticated ...

Quantitative Analysis of Random Migration of Cells Using Time-lapse Video Microscopy

Cell migration is a dynamic process, which is important for embryonic development, tissue repair, immune system function, and tumor invasion 1, 2. During directional migration, cells move rapidly in response to an extracellular chemotactic signal, or in response to intrinsic cues 3 provided by the basic motility machinery. Random migration occurs when a cell possesses low intrinsic directionality, allowing the cells to explore their local environment.
Cell migration is a complex process, in the initial response cell undergoes polarization and extends protrusions in the direction of migration 2. Traditional methods to measure migration such as the Boyden chamber migration assay is an easy method to measure chemotaxis in vitro, which allows measuring migration as an end point result. However, this approach neither allows measurement of individual migration parameters, nor does it allow to visualization of morphological changes that cell undergoes during migration.
Here, we present a method that allows us to monitor migrating cells in real time using video - time lapse microscopy. Since cell migration and invasion are hallmarks of cancer, this method will be applicable in studying cancer cell migration and invasion in vitro. Random migration of platelets has been considered as one of the parameters of platelet function 4, hence this method could also be helpful in studying platelet functions. This assay has the advantage of being rapid, reliable, reproducible, and does not require optimization of cell numbers. In order to maintain physiologically suitable conditions for cells, the microscope is equipped with CO2 supply and temperature thermostat. Cell movement is monitored by taking pictures using a camera fitted to the microscope at regular intervals. Cell migration can be calculated by measuring average speed and average displacement, which is calculated by Slidebook software.

This method allows monitoring of cells in real time and quantitative measurements of different cell migration parameters such as speed, displacement, and velocity. Unlike the traditional methods, this real time approach is not based on endpoint quantitative migration measurements; instead it allows monitoring and calculating different parameters continuously.

Time-lapse video Mikroskop ile Hücrelerinin Rastgele Göç Kantitatif Analiz

Cell migration is a dynamic process, which is important for embryonic development, tissue repair, immune system function, and tumor invasion 1, 2. During ...

Visualization of Craniofacial Development in the sox10: kaede Transgenic Zebrafish Line Using Time-lapse Confocal Microscopy

Vertebrate palatogenesis is a highly choreographed and complex developmental process, which involves migration of cranial neural crest (CNC) cells, convergence and extension of facial prominences, and maturation of the craniofacial skeleton. To study the contribution of the cranial neural crest to specific regions of the zebrafish palate a sox10: kaede transgenic zebrafish line was generated. Sox10 provides lineage restriction of the kaede reporter protein to the neural crest, thereby making the cell labeling a more precise process than traditional dye or reporter mRNA injection. Kaede is a photo-convertible protein that turns from green to red after photo activation and makes it possible to follow cells precisely. The sox10: kaede transgenic line was used to perform lineage analysis to delineate CNC cell populations that give rise to maxillary versus mandibular elements and illustrate homology of facial prominences to amniotes. This protocol describes the steps to generate a live time-lapse video of a sox10: kaede zebrafish embryo. Development of the ethmoid plate will serve as a practical example. This protocol can be applied to making a time-lapse confocal recording of any kaede or similar photoconvertible reporter protein in transgenic zebrafish. Furthermore, it can be used to capture not only normal, but also abnormal development of craniofacial structures in the zebrafish mutants.

Visualization of experimental data has become a key element in presenting results to the scientific community. Generation of live time-lapse recording of growing embryos contributes to better presentation and understanding of complex developmental processes. This protocol is a step-by-step guide to cell labeling via photoconversion of kaede protein in zebrafish.

Sox10 içinde Kraniyofasiyal Kalkınma Görselleştirme: kaede Transgenik Zebra balığı Hattı Time-lapse konfokal mikroskopi kullanılarak

Vertebrate palatogenesis is a highly choreographed and complex developmental process, which involves migration of cranial neural crest (CNC) cells, ...

Methods to Assess Subcellular Compartments of Muscle in C. elegans

Muscle is a dynamic tissue that responds to changes in nutrition, exercise, and disease state. The loss of muscle mass and function with disease and age are significant public health burdens. We currently understand little about the genetic regulation of muscle health with disease or age. The nematode C. elegans is an established model for understanding the genomic regulation of biological processes of interest. This worm&#8217;s body wall muscles display a large degree of homology with the muscles of higher metazoan species. Since C. elegans is a transparent organism, the localization of GFP to mitochondria and sarcomeres allows visualization of these structures in vivo. Similarly, feeding animals cationic dyes, which accumulate based on the existence of a mitochondrial membrane potential, allows the assessment of mitochondrial function in vivo. These methods, as well as assessment of muscle protein homeostasis, are combined with assessment of whole animal muscle function, in the form of movement assays, to allow correlation of sub-cellular defects with functional measures of muscle performance. Thus, C. elegans provides a powerful platform with which to assess the impact of mutations, gene knockdown, and/or chemical compounds upon muscle structure and function. Lastly, as GFP, cationic dyes, and movement assays are assessed non-invasively, prospective studies of muscle structure and function can be conducted across the whole life course and this at present cannot be easily investigated in vivo in any other organism.

Methods to Assess Subcellular Compartments of Muscle in C. elegans

Skeletal muscle is essential for locomotion and is the bodies&#8217; main protein store. Muscle health measurements within C. elegans are described. Prospective changes to muscle structure and function are assessed using localized GFP and cationic dyes.

Yöntemler içinde Muscle hücrealtı Kompartmanları DeğerlendirecekÜSKÜP<em&gt; ° C. elegans</em

Muscle is a dynamic tissue that responds to changes in nutrition, exercise, and disease state. The loss of muscle mass and function with disease and age ...