Biz Yaşam Bilimleri ve bu iş için fon Kraliçe&#39;nin Üniversitesi Sanat ve Bilim Fakültesi teşekkür ederim. Yanı sıra, biz gerekli çözeltiler, ekipman ve teknik destek sağlamak için Chin-Sang laboratuvar teşekkür ederim. Ayrıca tartışmaya katkılarından dolayı O, özellikle Tony, QGEM 2.011 teşekkür ederim.

1. Worms hazırlanması / yıkama <ol><li> Genç erişkin 11 solucanlar senkronize. </li><li> Bir 5 cm Kemotaksis plaka üzerine S Bazal Pipet 2 ml sadece OP50 E. çim temizlendiğini solucanlar düzenledi coli. Solucanlar tampona plaka yüzeyinden yıkanır sağlamak için gerektiği gibi plaka yatırın. </li><li> Bir mikrosantrifüj tüp içine solucan-S Bazal çözüm pipet 1 ml. </li><li> 10 sn 6.600 rpm bir picofuge kullanmak için santrifüj. </li><li> Rahatsız solucanlar pelet bırakarak, S Bazal aspire. </li><li> Solucanlar yıkamak için birkaç kez ters, mikrosantrifüj tüpüne 1 ml S Bazal çözüm ekleyin. </li><li> Adımları 1,4-1,6 başka üç kez tekrarlayın. </li><li> 10 saniye, beşinci kez, yaklaşık bir nihai hacme süpernatan aspire bu süre santrifüjleyin. 100 ul. </li><li> Her 2 ul örnek 50 ila 250 solucanlar olmadığından emin olmak için bir NGM plaka üzerine solucanlar Pipet 2 ul. AyarlamakG Bazal daha küçük ya da daha büyük hacimde solucanlar yeniden süspansiyona tabi olarak, S Bazal solucanlar konsantrasyonu. </li><li> Hemen 1 saat için solucan yıkandıktan sonra tahlilinde solucanlar kullanın. </li></ol> 2. Test Tabaklar hazırlanması <ol><li> 5 cm plakanın alt işaretleme, 4 eşit parçaya bölünür çanak bölmek. Kemotaksis NGM agar veya kullanılabilir. 3 çalışmaların en az test edilen genetik gerginlik başına gerekmektedir. </li><li> Kökeni etrafında yarıçapı 0.5 cm (Şekil 1) bir daire işaretleyin. </li><li> &quot;Test&quot; veya siteleri kökeni ve birbirinden eşit uzaklıkta olmasını sağlamak için bir &quot;C&quot; &quot;Kontrol,&quot; için ya bir &quot;T&quot; ile her kadranda bir nokta işaretleyin. Noktaları en az 2 cm uzakta kökenli olmalıdır. Testi kadran ve sağ üst ve kontrol grubu olarak sol alt kadran (Şekil 1) olarak sol üst ve sağ alt kadran işaretleyin. </li></ol> 3. Testi Koşu </p&gt; <ol><li> Test bileşiği ve 0.5 M azit (bir anestezik bir kadran erişme üzerine solucanlar tutmak için kullanılan), eşit hacimlerde birleştirerek test çözeltisi karıştırın. 0.5 M azid ile, test bileşiğinin seyreltmek için kullanılan çözücü ile birleştirilmesi ile kontrol solüsyonu karıştırın. </li><li> Kalkış üzerine pelet solucan çözeltisi (1.8 &#39;de hazırlanan) (iki satır kesiştiği) Pipet 2 ul. </li><li> Iki &quot;T&quot; siteleri üzerine test çözeltisinin hemen sonra, pipet 2 ul. Benzer şekilde, iki &quot;C&quot; siteleri üzerine kontrol solüsyonu ile aynı miktarda pipetle. </li><li> Solucan ve odorant damla agar emilir edildikten sonra, kapakları yerine ve plakalar ters. </li><li> 60 dakika sonra, 4 ° C inkübatör solucanlar yerleştirin. Bu sayılabilir yalnızca inkübatör bir tabak çıkarın. Oda sıcaklığında solucanlar bırakarak onları tekrar harekete izin verebilir. </li><li> Tamamen iç Ortam geçti her kadranda solucan sayısını kaydedinle. </li><li> Test ve kontrol kadran hem de kontrol çözeltisi kullanılarak adımları 3,2-3,6 tekrarlayın. Bu kontrol plaka olarak görev yapacak. Üç tür plakalar yapılmış ve uygun bir kontrol olarak hizmet çalıştırılmalıdır. </li></ol> 4. Skorlar Sözlü / Kemotaksis Endeks belirlenmesi <ol><li> Denklem 1 kullanarak kemotaksis indeksi hesaplayın. Bu -1.0 ve +1.0 arasında bir kemotaktik indeksi verecektir. </li></ol> (1) Kemotaksis İndeksi = (Test bölgeleri Her iki içerisinde Worms - Her iki kontrol bölgeleri içerisinde Worms) / (Attı Worms # Toplam) Bir +1.0 puan hedefe doğru maksimum çekim gösterir ve kimyasal hedef içeren kadran gelen solucanlar% 100 temsil eder. -1.0 Bir dizin maksimum itme kanıtıdır. Benzer test yöntemleri zaten 3 (Tablo 1) istihdam edilmiştir. <ol start="2"><li> Ortalama Kemotaksi rapors Endeksi (CI) ve standart hata (SEM) anlamına gelir. Öğrenci T-testi test ve kontrol plakaları verileri karşılaştırarak yapın. </li></ol>

<ol>
	<li>Bargmann, C. I., Hartwieg, E., Horvitz, H. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Odorant-selective+genes+and+neurons+mediate+olfaction+in+C.+elegans.">Odorant-selective genes and neurons mediate olfaction in C. elegans.</a> Cell. 74, 515-527 (1993).</li><li>Bargmann, C. I., Horvitz, H. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemosensory+neurons+with+overlapping+functions+direct+chemotaxis+to+multiple+chemicals+in+C.+elegans.">Chemosensory neurons with overlapping functions direct chemotaxis to multiple chemicals in C. elegans.</a> Neuron. 7, 729-742 (1991).</li><li>Lee, J., Jee, C., McIntire, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ethanol+preference+in+C.+elegans.">Ethanol preference in C. elegans.</a> Genes Brain Behav. 8, 578-585 (2009).</li><li>Swierczek, N. A., Giles, A. C., Rankin, C. H., Kerr, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-throughput+behavioral+analysis+in+C.+elegans.">High-throughput behavioral analysis in C. elegans.</a> Nat. Methods. 8, 592-598 (2011).</li><li>Ward, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemotaxis+by+the+nematode+Caenorhabditis+elegans:+identification+of+attractants+and+analysis+of+the+response+by+use+of+mutants.">Chemotaxis by the nematode Caenorhabditis elegans: identification of attractants and analysis of the response by use of mutants.</a> Proc. Natl. Acad. Sci. U.S.A. 70, 817-821 (1973).</li><li>Colbert, H. A., Bargmann, C. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Odorant-specific+adaptation+pathways+generate+olfactory+plasticity+in+C.+elegans.">Odorant-specific adaptation pathways generate olfactory plasticity in C. elegans.</a> Neuron. 14, 803-812 (1995).</li><li>Kauffman, A., Parsons, L., Stein, G., Wills, A., Kaletsky, R., Murphy, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=C.+elegans+Positive+Butanone+Learning,+Short-term,+and+Long-term+Associative+Memory+Assays.">C. elegans Positive Butanone Learning, Short-term, and Long-term Associative Memory Assays.</a> J. Vis. Exp. (49), e2490 (2011).</li><li>Troemel, E. R., Kimmel, B. E., Bargmann, C. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reprogramming+chemotaxis+responses:+sensory+neurons+define+olfactory+preferences+in+C.+elegans.">Reprogramming chemotaxis responses: sensory neurons define olfactory preferences in C. elegans.</a> Cell. 91, 161-169 (1997).</li><li>Pierce-Shimomura, J. T., Morse, T. M., Lockery, S. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+fundamental+role+of+pirouettes+in+Caenorhabditis+elegans+chemotaxis.">The fundamental role of pirouettes in Caenorhabditis elegans chemotaxis.</a> J Neurosci. 19, 9557-9569 (1999).</li><li>Frokjaer-Jensen, C., Ailion, M., Lockery, S. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ammonium-acetate+is+sensed+by+gustatory+and+olfactory+neurons+in+Caenorhabditis+elegans.">Ammonium-acetate is sensed by gustatory and olfactory neurons in Caenorhabditis elegans.</a> PLoS One. 3, e2467 (2008).</li><li>Porta-de-la-Riva, M., Fontrodona, L., Villanueva, A., Cer&#243;n, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Basic+Caenorhabditis+elegans+Methods:+Synchronization+and+Observation.">Basic Caenorhabditis elegans Methods: Synchronization and Observation.</a> J. Vis. Exp. (64), e4019 (2012).</li><li>Sengupta, P., Bargmann, C. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+fate+specification+and+differentiation+in+the+nervous+system+of+Caenorhabditis+elegans.">Cell fate specification and differentiation in the nervous system of Caenorhabditis elegans.</a> Dev. Genet. 18, 73-80 (1996).</li><li>Hart, A. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Behavior.">Behavior.</a> WormBook. , (1895).</li></ol>

Biz ifşa hiçbir şey yok.

Kemotaksis, nöronal ve hücresel mekanizmalar karmaşık bir dizi tarafından kontrol edilen, ancak kolay ve objektif kemotaksi deneyleri kullanılarak belirlenebilir. Testleri en iyi sonuçları elde etmek için, bazı kritik adımlar alınmalıdır. İlk olarak, solucanlar evreleme tutarlı deneysel sonuçlar veren esastır. Farklı yaşam aşamalarında Worms farklı 13 davranırlar, bu yüzden karışık aşamada solucanlar olabilir çarpık deneysel sonuçlar. İkinci olarak, tüm E. sağlanmas...

Ward, 1973 5 kemotaksis testi geliştirdi, ve o zamandan beri geniş kapsamlı uygulamalar olmuştur. Nörobiyoloji kemotaksi testleri kullanarak çeşitli yararlanmıştır olan bir alandır. Koku uyum, öğrenme ve bellek basit bir formu, C. gösterilmiştir kemotaksi testleri 6 kullanarak elegans. Ayrıca göstermek için kullanılmış olduğunu ° C. elegans etanol solucanlar davranışsal plastisite gösterir sadece hoşgörü-bir sonucu gelişebilir, ama bu da solucanlar insanlarda 3 alkol bağımlılığının çalışmada çok yararlı olabilir göstermektedir. Tahliller da C yeteneğini göstermek için geliştirilmiştir elegans dernekler chemoattractants ve gıda (OP50) 7 arasındaki solucanlar tarafından yapılmış olduğunu göstererek kısa ve uzun süreli hafıza depolamak için. Ayrıca C, ilgili mevcut geniş bilgi verilir elegans genomunun, KemotaksiC. s davranış elegans mutasyonlar 1,8 uyararak defalarca değiştirilmiş. Bu, C. geliştirilmesi gibi birçok heyecan verici mühendislik olanakları, sağlar bir biyoremidasyon aracı olarak elegans. Böylece, 1973 yılında kemotaksis testin ilk geliştirme yana, sık sık değişmiş ve disiplinlerden çeşitli gizemleri aydınlatmak için kullanılmıştır. Bazı testler bir hedefe doğru solucanlar tarafından alınan belirli bir rota keşfetmek amaçladık. Bu tür prototip testi Ward 5 tarafından geliştirilmiştir. Üç solucanlar 15 dakika boyunca erimiş agar üzerine yerleştirilmiştir. Onların hareketleri onlar plakasının merkezinde bir çekici bir degrade kadar plaka çevre gitti onlar sol baskı tarafından takip edildi. Plaka üzerinde tüm solucanlar her deneme sonunda kloroform kullanılarak gözaltına alındı. Bu yöntemin bir alt çekici ve kontrol ile bir levhanın ortasında bir tek solucan yerleştirilmiştirkökeni 2 t eşit ve zıt mesafelerde. Pierce-Shimomura&#39;nın ve ark. Kemotaksis 9 dahil hareketinin tam doğası gözlemlemek için bir analiz geliştirdik. Bağımsız solucanlar çekici madde kaynağı ile sonuçlanan, 9 cm Petri tabakları çekici veya radyal şekilli degrade bir konsantrasyonunu ihtiva eden ya da yerleştirilmiştir. Solucan tanınan bir bilgisayar yazılım programı gözlenen davranışı kaydetmek için kullanıldı. Mikroskop bağlı bir video kamera tahlil solucan görüş alanında kaldı sağlamak için koştu otomatik olarak Petri kabı ayarlamak için sahne ile birlikte çalıştı. Buradan, daha ayrıntılı bilgi C. tarafından görüntülenen pirouettes nedeni ile ilgili keşfedildi elegans. Burada açıklanan daha yakın bir şekilde diğer analizler, test bileşikleri için solucanlar büyük bir nüfusun yanıt olarak test edilmiştir. İki çeyrek kemotaksi deneyleri olmuşturÇeşitli nöronlar, reseptörler, ve sinyal iletim molekülleri, C &#39;oynadığı roller keşfetmek için kullanılan elegans çeşitli bileşikler 1 maruz kalmıştır. 20-50 arasında yıkanır solucanlar, anestetik, sodyum azit (NaN3) birlikte polar ucunda bir çekici ve bir kontrol plakasının merkezine yakın yerleştirilmiştir. 60 dakika sonra, -1.0 den +1.0 değerleri ile bir kemotaktik indeksi cezbedici veya kontrol yapıştırılmış kaç solucan arasındaki fark esas alınarak oluşturulmuştur. Daha önce tahlil katı non-motil solucanlar değerlendirmek için başarısız olsa da benzer bir kemotaktik indeksi, bu makalede rapor tahlilinde kullanılmıştır. Bu deney, daha sonra daha fazla kemotaksisidir nöronal ablasyon etkilerini test uygulandı. 100-200 solucanlar dört kadran 3 içeren bir tabak ortasına yerleştirildi nerede söz konusu testin başka bir varyasyonu yapıldı. Bitişik çeyrek daireler ya ya da test yermadde kontrol. Önceki deneylerde olduğu gibi, solucanlar atılan önce sodyum azid hareketi ile hareketsizleştirildi. Benzer bir yöntem, C cevabı değerlendirmek için bir yol olarak burada açıklanan çeşitli bileşikler için elegans. Ancak, aşağıdaki yöntemi yalnızca hareketsiz solucanlardan mobil ayıran bir eşik mesafe geçti solucanlar değerlendirilmesi yararı vardır. Diğer testler hareketsiz solucanlar görmezden için benzer yönergeleri dahil etmişlerdir. Frøkjær-Jensen ve diğerleri. Uçucu ve su hem de çözünebilir bileşikler 10 test etmek için kullanılabilecek bir analiz geliştirdik yönlü. Bir Petri kabı dört parçaya bölündü. Üst ve alt çeyrek solvent içermemektedir. Sol kadran su içerdiği ve doğru cezbedici içeriyordu. Suda çözünen bileşikler aga doğrudan yerleştirildi ise uçucu koku test ederken, analit, uygun kadran üzerinde, çanak kapağı yerleştirildir. C. kemotaktik tepkiyi değerlendirmek için şu anda var olan yöntemler elegans sürekli kullanımı, verimlilik ve doğruluk kolaylığı optimize etmek için rafine edilmektedir. Burada açıklanan tahlil solucanlar en fazla (. Maksimum verim: 250 solucanlar / plaka başına saat, Lee ve ark 3 gösterdiği verim biraz daha fazla) değerlendirmek yeteneğine sahipken Yani,, bu yöntemin gerçek gücü olan Daha önce testleri (Tablo 1) özelliklerini birçok kısa doruk noktası.

<table border="1">
 <tbody>
 <tr>
 <td>Name of Reagent/Material</td>
 <td>Company</td>
 <td>Catalogue Number</td>
 <td>Comments</td>
 </tr>
 <tr>
 <td>S Basal (- cholesterol) [5.8 g NaCl; 1 M K phosphate buffer, pH 6.0; dH2O to 1 liter.] Autoclave</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>PicoFuge</td>
 <td>Stratagene</td>
 <td>400552</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Microscopes</td>
 <td>Leica</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Dissecting</td>
 <td>Leica</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>P1000 Pipette</td>
 <td>Gilson</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>P10 Pipette</td>
 <td>Gilson</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>0.5 M Sodium Azide</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Chemotaxis Agar [1.6% BBL-agar (Benton-Dickinson) or 2% Difco-agar. Autoclave. Add 5 mM potassium phosphate, pH 6.0; 1 mM CaCl2, 1 mM MgSO4]</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Ethanol/Distilled Water</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Test Compounds (eg. 0.5% diacetyl)</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Agar</td>
 <td>Bio-Rad</td>
 <td>166-0600</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>NH4Cl</td>
 <td>Amresco</td>
 <td>CA97062-046</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>MOPS</td>
 <td>VWR</td>
 <td>CA12001-120</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>NH4OH</td>
 <td>BDH</td>
 <td>CABDH8641-2</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>0.25% Tween 20</td>
 <td>Bio-Rad</td>
 <td>170-6531</td>
 <td>&nbsp;</td>
 </tr>
 </tbody>
</table>

c. elegans chemotaxis assay

Birçok organizma kemotaksis, gıda kaynakları bulmak zararlı maddeler önlemek ve arkadaşları bulmak için kullanın. Caenorhabditis elegans etkileyici kemotaksis davranış vardır. Bir odorant için solucanlar yanıtı test arkasında öncül bir alanda koyun ve bir odorant yanıt olarak uyarılmış hareketi gözlemlemektir. Hatta mevcut olan birçok deneyleri ile, önemli örnek boyutu korunarak birbirleriyle solucanlar etkileşimi en aza indirirken, kontrol ve test alanları hem de göreli konumu başlangıç ​​solucan optimize ilerleme 1-10 bir çalışma devam etmektedir. Burada anlatılan yöntemi Bargmann ve ark. 1. tarafından geliştirilen test değiştirerek bu konuları ele amaçlamaktadır. Bir Petri kabı dört parçaya bölünür, &quot;Test&quot; ve iki işaretli iki zıt kadran &quot;Kontrol&quot; belirlenir. Anestezik tüm test ve kontrol sitelerinin yerleştirilir. Solucanlar bir Circl ile plaka ortasına yerleştirilire non-motil solucanlar yok sayılır sağlamak için kökenli etrafında oldu. Dört kadran sistemi kullanılarak yerine bir 2 ya da iki 1 bakılmaksızın başladı kökeni hangi tarafında, onlar test ve kontrol örnekleri eşit uzaklıkta olduğu gibi, solucanlar hareketi önyargı ortadan kaldırır. Bu solucan gecikme ya da amaçlanan yolun yanlış yorumlanması verimli, daha dolambaçlı yol almaya zorlayabilir bir odorant, cevap diğer solucanlar bir küme ile seyahat etmek zorunda olan solucanlar sorunu circumvents. Bu yöntem aynı zamanda daha büyük bir örneklem büyüklüğü olan ve araştırmacı katılımsız tahlil çalıştırın ve ayrılan zaman süresi bitiminde solucanlar puan için izin vererek pratik avantajlar gösterir.

Yabani tip (N2) C Karşılaştırılması ODR-10 (KY10) mutant için elegans. Biz, bilinen bir C diasetil el elegans kemoatraktan, diasetil 1,12 için reseptör olmayan bir mutantı olduğu için vahşi tip solucanlar karşılaştırma. Doğal tipte (N2) solucanlar için kemotaktik indeksi etanol ve 0,839-0,100 ± 0.066 idi ± 0,031-0,5% diasetil. Beklendiği gibi, diasetil yabani tip solucanlar (p &lt;0.003) anlamlı bir chemoatt...

Watch this Scientific Journal Video about C. elegans Chemotaxis Assay at JoVE.com

C. elegans Chemotaxis Assay

Niceliksel kemotaktik tepkiyi değerlendirmek için bir yöntem<em&gt; Caenorhabditis elegans</em&gt; Tarif edilmektedir. Bir kemotaktik indeksi (CI) tam olarak belli hedeflere solucanlar yanıtı değerlendirmek ve suşları ve ilgi bileşikler arasında karşılaştırma bir platform olarak hizmet için bir yol olarak kullanılmıştır.

C. elegans Kemotaksis Deneyi

Many organisms use chemotaxis to seek out food sources, avoid noxious substances, and find mates.  Caenorhabditis elegans has impressive chemotaxis ...

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C. elegans Chemotaxis Assay

45.5K Görüntüleme.  Queen's University. Niceliksel kemotaktik tepkiyi değerlendirmek için bir yöntem Caenorhabditis elegans Tarif edilmektedir. Bir kemotaktik indeksi (CI) tam olarak belli hedeflere solucanlar yanıtı değerlendirmek ve suşları ve ilgi bileşikler arasında karşılaştırma bir platform olarak hizmet için bir yol olarak kullanılmıştır.

Video: C. elegans Chemotaxis Assay

Generation of Stable Transgenic C. elegans Using Microinjection

Transgenic Caenorhabditis elegans can be readily created via microinjection of a DNA plasmid solution into the gonad 1. The plasmid DNA rearranges to form extrachromosomal concatamers that are stably inherited, though not with the same efficiency as actual chromosomes 2. A gene of interest is co-injected with an obvious phenotypic marker, such as rol-6 or GFP, to allow selection of transgenic animals under a dissecting microscope. The exogenous gene may be expressed from its native promoter for cellular localization studies. Alternatively, the transgene can be driven by a different tissue-specific promoter to assess the role of the gene product in that particular cell or tissue. This technique efficiently drives gene expression in all tissues of C. elegans except for the germline or early embryo 3. Creation of transgenic animals is widely utilized for a range of experimental paradigms. This video demonstrates the microinjection procedure to generate transgenic worms. Furthermore, selection and maintenance of stable transgenic C. elegans lines is described.

This video demonstrates the technique of microinjection into the gonad of C. elegans to create transgenic animals.

Mikroenjeksiyon Kararlı Transgenik C. elegans Üretimi

Transgenic Caenorhabditis elegans can be readily created via microinjection of a DNA plasmid solution into the gonad 1.  The plasmid DNA rearranges to ...

Biyoloji

Application of a C. elegans Dopamine Neuron Degeneration Assay for the Validation of Potential Parkinson's Disease Genes

Improvements to the diagnosis and treatment of Parkinson's disease (PD) are dependent upon knowledge about susceptibility factors that render populations at risk. In the process of attempting to identify novel genetic factors associated with PD, scientists have generated many lists of candidate genes, polymorphisms, and proteins that represent important advances, but these leads remain mechanistically undefined. Our work is aimed toward significantly narrowing such lists by exploiting the advantages of a simple animal model system. While humans have billions of neurons, the microscopic roundworm Caenorhabditis elegans has precisely 302, of which only eight produce dopamine (DA) in hemaphrodites. Expression of a human gene encoding the PD-associated protein, alpha-synuclein, in C. elegans DA neurons results in dosage and age-dependent neurodegeneration.
Worms expressing human alpha-synuclein in DA neurons are isogenic and express both GFP and human alpha-synuclein under the DA transporter promoter (Pdat-1). The presence of GFP serves as a readily visualized marker for following DA neurodegeneration in these animals. We initially demonstrated that alpha-synuclein-induced DA neurodegeneration could be rescued in these animals by torsinA, a protein with molecular chaperone activity 1. Further, candidate PD-related genes identified in our lab via large-scale RNAi screening efforts using an alpha-synuclein misfolding assay were then over-expressed in C. elegans DA neurons. We determined that five of seven genes tested represented significant candidate modulators of PD as they rescued alpha-synuclein-induced DA neurodegeneration 2. Additionally, the Lindquist Lab (this issue of JoVE) has performed yeast screens whereby alpha-synuclein-dependent toxicity is used as a readout for genes that can enhance or suppress cytotoxicity. We subsequently examined the yeast candidate genes in our C. elegans alpha-synuclein-induced neurodegeneration assay and successfully validated many of these targets 3, 4.
Our methodology involves generation of a C. elegans DA neuron-specific expression vector using recombinational cloning of candidate gene cDNAs under control of the Pdat-1 promoter. These plasmids are then microinjected in wild-type (N2) worms, along with a selectable marker for successful transformation. Multiple stable transgenic lines producing the candidate protein in DA neurons are obtained and then independently crossed into the alpha-synuclein degenerative strain and assessed for neurodegeneration, at both the animal and individual neuron level, over the course of aging.

This video demonstrates how to use C. elegans to assess dopaminergic neuron neurodegeneration as a model for Parkinson's disease. Furthermore, genetic screens are used to identify factors that either enhance degeneration or are neuroprotective.

Potansiyel Parkinson Hastalığı Genler Doğrulama için C. elegans Dopamin Nöron Dejenerasyonu Testi Uygulama

Improvements to the diagnosis and treatment of Parkinson's disease (PD) are dependent upon knowledge about susceptibility factors that render populations ...

Paradigms for Pharmacological Characterization of C. elegans Synaptic Transmission Mutants

The nematode, Caenorhabditis elegans, has become an expedient model for studying neurotransmission. C. elegans is unique among animal models, as the anatomy and connectivity of its nervous system has been determined from electron micrographs and refined by pharmacological assays. In this video, we describe how two complementary neural stimulants, an acetylcholinesterase inhibitor, called aldicarb, and a gamma-aminobutyric acid (GABA) receptor antagonist, called pentylenetetrazole (PTZ), may be employed to specifically characterize signaling at C. elegans neuromuscular junctions (NMJs) and facilitate our understanding of antagonistic neural circuits.
Of 302 C. elegans neurons, nineteen GABAergic D-type motor neurons innervate body wall muscles (BWMs), while four GABAergic neurons, called RMEs, innervate head muscles. Conversely, thirty-nine motor neurons express the excitatory neurotransmitter, acetylcholine (ACh), and antagonize GABA transmission at BWMs to coordinate locomotion. The antagonistic nature of GABAergic and cholinergic motor neurons at body wall NMJs was initially determined by laser ablation and later buttressed by aldicarb exposure. Acute aldicarb exposure results in a time-course or dose-responsive paralysis in wild-type worms. Yet, loss of excitatory ACh transmission confers resistance to aldicarb, as less ACh accumulates at worm NMJs, leading to less stimulation of BWMs. Resistance to aldicarb may be observed with ACh-specific or general synaptic function mutants. Consistent with antagonistic GABA and ACh transmission, loss of GABA transmission, or a failure to negatively regulate ACh release, confers hypersensitivity to aldicarb. Although aldicarb exposure has led to the isolation of numerous worm homologs of neurotransmission genes, aldicarb exposure alone cannot efficiently determine prevailing roles for genes and pathways in specific C. elegans motor neurons. For this purpose, we have introduced a complementary experimental approach, which uses PTZ.
Neurotransmission mutants display clear phenotypes, distinct from aldicarb-induced paralysis, in response to PTZ. Wild-type worms, as well as mutants with specific inabilities to release or receive ACh, do not show apparent sensitivity to PTZ. However, GABA mutants, as well as general synaptic function mutants, display anterior convulsions in a time-course or dose-responsive manner. Mutants that cannot negatively regulate general neurotransmitter release and, thus, secrete excessive amounts of ACh onto BWMs, become paralyzed on PTZ. The PTZ-induced phenotypes of discrete mutant classes indicate that a complementary approach with aldicarb and PTZ exposure paradigms in C. elegans may accelerate our understanding of neurotransmission. Moreover, videos demonstrating how we perform pharmacological assays should establish consistent methods for C. elegans research.

This video demonstrates how to employ two neural stimulants, aldicarb and pentylenetetrazole (PTZ), in complementary ways to study synaptic function in the nematode, C. elegans. This complementary approach may also be used to shed light on evolutionarily conserved mechanisms for modulating neuronal synchrony and has implications for epilepsy and seizures.

C. elegans Sinaptik İletim Mutants farmakolojik karakterizasyonu için Paradigmalar

The nematode, Caenorhabditis elegans, has become an expedient model for studying neurotransmission. C. elegans is unique among animal models, as the ...

Dissecting and Recording from The C. Elegans Neuromuscular Junction

Neurotransmission is the process by which neurons transfer information via chemical signals to their post-synaptic targets, on a rapid time scale. This complex process requires the coordinated activity of many pre- and post-synaptic proteins to ensure appropriate synaptic connectivity, conduction of electrical signals, targeting and priming of secretory vesicles, calcium sensing, vesicle fusion, localization and function of postsynaptic receptors and finally, recycling mechanisms. As neuroscientists it is our goal to elucidate which proteins function in each of these steps and understand their mechanisms of action. Electrophysiological recordings from synapses provide a quantifiable read out of the underlying electrical events that occur during synaptic transmission. By combining this technique with the powerful array of molecular and genetic tools available to manipulate synaptic proteins in C. elegans, we can analyze the resulting functional changes in synaptic transmission. 
The C. elegans NMJs formed between motor neurons and body wall muscles control locomotion, therefore, mutants with uncoordinated locomotory phenotypes (known as unc s) often perturb synaptic transmission at these synapses 1. Since unc mutants are maintained on a rich supply of a bacterial food source, they remain viable as long as they retain some pharyngeal pumping ability to ingest food. This, together with the fact that C. elegans exist as hermaphrodites, allows them to pass on mutant progeny without the need for elaborate mating behaviors. These attributes, coupled with our recent ability to record from the worms NMJs 2,3,7 make this an excellent model organism in which to address precisely how unc mutants impact neurotransmission. 
The dissection method involves immobilizing adult worms using a cyanoacrylic glue in order to make an incision in the worm cuticle exposing the NMJs. Since C. elegans adults are only 1 mm in length the dissection is performed with the use of a dissecting microscope and requires excellent hand-eye coordination. NMJ recordings are made by whole-cell voltage clamping individual body wall muscle cells and neurotransmitter release can be evoked using a variety of stimulation protocols including electrical stimulation, light-activated channel-rhodopsin-mediated depolarization 4 and hyperosmotic saline, all of which will be briefly described.

Application of electrophysiology to accessible synapses provides a quantifiable measure of synaptic activity, useful in analyzing synaptic mutants. This article describes a dissection method used to expose the neuromuscular junctions (NMJ) of Caenorhabditis elegans (C. elegans) and briefly discusses some of the uses to which this preparation can be applied.

C. Elegans Nöromüsküler Kavşağı Diseksiyon ve kayıt

Neurotransmission is the process by which neurons transfer information via chemical signals to their post-synaptic targets, on a rapid time scale.   This ...

Generation of Transgenic C. elegans by Biolistic Transformation

The number of laboratories using the free living nematode C. elegans is rapidly growing. The popularity of this biological model is attributed to a rapid generation time and short life span, easy and inexpensive maintenance, fully sequenced genome, and array of RNAi resources and mutant animals. Additionally, analysis of the C. elegans genome revealed a great similarity between worms and higher vertebrates, which suggests that research in worms could be an important adjunct to studies performed in whole mice or cultured cells. A powerful and important part of worm research is the ability to use transgenic animals to study gene localization and function. Transgenic animals can be created either via microinjection of the worm germline or through the use of biolistic bombardment. Bombardment is a newer technique and is less familiar to a number of labs. Here we describe a simple protocol to generate transgenic worms by biolistic bombardment with gold particles using the Bio-Rad PDS-1000 system. Compared with DNA microinjection into hermaphrodite germline, this protocol has the advantage of not requiring special skills from the operator with regards to identifying worm anatomy or performing microinjection. Further multiple transgenic lines are usually obtained from a single bombardment. Also in contrast to microinjection, biolistic bombardment produces transgenic animals with both extrachromosomal arrays and integrated transgenes. The ability to obtain integrated transgenic lines can avoid the use of mutagenic protocols to integrate foreign DNA. In conclusion, biolistic bombardment can be an attractive method for the generation of transgenic animals, especially for investigators not interested in investing the time and effort needed to become skilled at microinjection.

Generation of Transgenic C. elegans by Biolistic Transformation

Transgenic worms are commonly used in C. elegans research. Described is a simple, yet effective, protocol to introduce transgenes into worms using biolistic bombardment with DNA-coated gold particles. The effort involved and results of bombardment compare favorably with microinjection for the generation of transgenic animals.

Transgenik, Üretimi C. elegans Biyolistik Dönüşüm

The number of laboratories using the free living nematode C. elegans is rapidly growing. The popularity of this biological model is attributed to a rapid ...

High-throughput Screening and Biosensing with Fluorescent C. elegans Strains

High-throughput screening (HTS) is a powerful approach for identifying chemical modulators of biological processes. However, many compounds identified in screens using cell culture models are often found to be toxic or pharmacologically inactive in vivo1-2. Screening in whole animal models can help avoid these pitfalls and streamline the path to drug development.
C. elegans is a multicellular model organism well suited for HTS. It is small (&lt;1 mm) and can be economically cultured and dispensed in liquids. C. elegans is also one of the most experimentally tractable animal models permitting rapid and detailed identification of drug mode-of-action3.
We describe a protocol for culturing and dispensing fluorescent strains of C. elegans for high-throughput screening of chemical libraries or detection of environmental contaminants that alter the expression of a specific gene. Large numbers of developmentally synchronized worms are grown in liquid culture, harvested, washed, and suspended at a defined density. Worms are then added to black, flat-bottomed 384-well plates using a peristaltic liquid dispenser. Small molecules from a chemical library or test samples (e.g., water, food, or soil) can be added to wells with worms. In vivo, real-time fluorescence intensity is measured with a fluorescence microplate reader. This method can be adapted to any inducible gene in C. elegans for which a suitable reporter is available. Many inducible stress and developmental transcriptional pathways are well defined in C. elegans and GFP transgenic reporter strains already exist for many of them4. When combined with the appropriate transgenic reporters, our method can be used to screen for pathway modulators or to develop robust biosensor assays for environmental contaminants. 
We demonstrate our C. elegans culture and dispensing protocol with an HTS assay we developed to monitor the C. elegans cap &#x2018;n&#x2019; collar transcription factor SKN-1. SKN-1 and its mammalian homologue Nrf2 activate cytoprotective genes during oxidative and xenobiotic stress5-10. Nrf2 protects mammals from numerous age-related disorders such as cancer, neurodegeneration, and chronic inflammation and has become a major chemotherapeutic target11-13.Our assay is based on a GFP transgenic reporter for the SKN-1 target gene gst-414, which encodes a glutathione-s transferase6. The gst-4 reporter is also a biosensor for xenobiotic and oxidative chemicals that activate SKN-1 and can be used to detect low levels of contaminants such as acrylamide and methyl-mercury15-16.

High-throughput Screening and Biosensing with Fluorescent C. elegans Strains

A procedure for liquid-based culturing and dispensing of C. elegans strains expressing fluorescent reporter proteins is described that does not require expensive sorting equipment. This approach can be applied to numerous inducible C. elegans genes for drug discovery or biosensing of contaminants.

Floresan ile yüksek verim Tarama ve Biyoalgılayıcı C. elegans Suşlarının

High-throughput screening (HTS) is a powerful approach for identifying chemical modulators of biological processes.  However, many compounds identified in ...

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 ...

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 ...

A Method for Culturing Embryonic C. elegans Cells

C. elegans is a powerful model system, in which genetic and molecular techniques are easily applicable. Until recently though, techniques that require direct access to cells and isolation of specific cell types, could not be applied in C. elegans. This limitation was due to the fact that tissues are confined within a pressurized cuticle which is not easily digested by treatment with enzymes and/or detergents. Based on early pioneer work by Laird Bloom, Christensen and colleagues 1 developed a robust method for culturing C. elegans embryonic cells in large scale. Eggs are isolated from gravid adults by treatment with bleach/NaOH and subsequently treated with chitinase to remove the eggshells. Embryonic cells are then dissociated by manual pipetting and plated onto substrate-covered glass in serum-enriched media. Within 24 hr of isolation cells begin to differentiate by changing morphology and by expressing cell specific markers. C. elegans cells cultured using this method survive for up 2 weeks in vitro and have been used for electrophysiological, immunochemical, and imaging analyses as well as they have been sorted and used for microarray profiling.

A Method for Culturing Embryonic C. elegans Cells

We describe here a revised protocol for large-scale culture of embryonic C. elegans cells. Embryonic C. elegans cells cultured in vitro using this method, appear to differentiate and recapitulate the expression of genes in a cell specific manner. Techniques that require direct access to the cells or isolation of specific cell types from the other tissues can be applied on C. elegans cultured cells.

Kültürleme Embriyonik için bir yöntem<em&gt; C. elegans</em&gt; Hücreler

C. elegans is a powerful model system, in which genetic and molecular techniques are easily applicable. Until recently though, techniques that require ...

Antibody Staining in C. Elegans Using &quot;Freeze-Cracking&quot;

To stain C. elegans with antibodies, the relatively impermeable cuticle must be bypassed by chemical or mechanical methods. "Freeze-cracking" is one method used to physically pull the cuticle from nematodes by compressing nematodes between two adherent slides, freezing them, and pulling the slides apart. Freeze-cracking provides a simple and rapid way to gain access to the tissues without chemical treatment and can be used with a variety of fixatives. However, it leads to the loss of many of the specimens and the required compression mechanically distorts the sample. Practice is required to maximize recovery of samples with good morphology. Freeze-cracking can be optimized for specific fixation conditions, recovery of samples, or low non-specific staining, but not for all parameters at once. For antibodies that require very hard fixation conditions and tolerate the chemical treatments needed to chemically permeabilize the cuticle, treatment of intact nematodes in solution may be preferred. If the antibody requires a lighter fix or if the optimum fixation conditions are unknown, freeze-cracking provides a very useful way to rapidly assay the antibody and can yield specific subcellular and cellular localization information for the antigen of interest.

Antibody Staining in C. Elegans Using "Freeze-Cracking"

"Freeze-cracking," a method for exposing the inner tissues of the nematode C. elegans to antibodies for protein localization, is demonstrated.

Antikor boyamasında<em&gt; C. Elegans</em&gt; &quot;Freeze-Cracking kullanma&quot;

To stain C.  elegans with antibodies, the relatively impermeable cuticle must be  bypassed by chemical or mechanical methods.  "Freeze-cracking" is one ...

C. elegans Kemotaksis Deneyi

Özet

Daha Fazla Video Keşfet

Bu videodaki bölümler

Mikroenjeksiyon Kararlı Transgenik C. elegans Üretimi

Potansiyel Parkinson Hastalığı Genler Doğrulama için C. elegans Dopamin Nöron Dejenerasyonu Testi Uygulama

C. elegans Sinaptik İletim Mutants farmakolojik karakterizasyonu için Paradigmalar

C. Elegans Nöromüsküler Kavşağı Diseksiyon ve kayıt

Transgenik, Üretimi C. elegans Biyolistik Dönüşüm

Floresan ile yüksek verim Tarama ve Biyoalgılayıcı C. elegans Suşlarının

Içinde Postembryonic Fenotiplerin Belirleme RNAi Tarama C. elegans

Temel

Kültürleme Embriyonik için bir yöntem

Antikor boyamasında