Biz çeşitli gelişimsel zaman noktalarında hayvan figürleri sağlamak için James Clark ederim. C. Bu çalışmada elegans suşları Araştırma Kaynakları için NIH Ulusal Merkezi (NCRR) tarafından desteklenen Caenorhabditis Genetik Merkezi, elde edilmiştir. Bu çalışma CUNY gelen JL ve CSD CIRG 1817 tarafından desteklenen ve NIH 1R15GM073678-01 ve CSD 1R15GM097692-01 tarafından Biz yazması üzerine yorumlar için Dr William J Rice, Dr Nathalia Holtzman ve Melissa Silvestrini ederim. Bu çalışmaları, New York Şehir Üniversitesi (S. Xiong) ve Graduate Center&#39;da bir doktora derecesi için gereksinimleri kısmen yerine getirilmesi yürütülmüştür.

1. Hedef Gen Dizi Defter RNAi Besleme Tabaklar hazırlanması <ol><li> Piyasada bulunan RNAi kütüphanelerin (örn. Kaynak BioScience Doğal Bilimler itibaren) kullanıyorsanız, 1.4 adıma geçin. Alternatif olarak, standart klonlama protokolü tarafından, vektör L4440, 8 RNAi plazmid yaygın olarak kullanılan bir solucan içine hedef gen dizileri klonu. </li><li> Bakterinin HT115 (DE3), 25 mcg / ml karbenisilin ile LB agar plaklarına kültürü onları ve 12.5 mcg / ml tetrasiklin içine rekombinant plazmid Transform, ve gelecekte kullanılmak üzere tek bir klon almak. </li><li> Bununla birlikte, deneyler için bir kontrol olarak kullanmak için bakteri suşu HT115 içine boş vektör L4440 dönüşümü. </li><li> 37 gece boyunca 100 ug / ml ampisilin ile 1 ml LB et suyu ° C. Kültür rekombinant ve boş hem de L4440 taşıyan bakteriler Tetrasiklin o RNAi randıman düşer olmasından dolayı eklenir. </li><li> Gecede cultu içine 100 mg / ml ampisilin ile başka bir 5 ml LB suyu ekleyinre, 37 başka bir 4-6 saat süreyle inkübe ° C </li><li> Tohum 0.5 ml rekombinant veya boş L4440 taşıyan RNAi solucanı plakalar üzerine bakteri kültürü. Klon adı ile tüm plakaları etiketleyin. </li><li> Plakaları işaretleyin ve 37 gece inkübe ° C bakteriyel çim büyümeye; Bu hedef gen sekansı olan veya olmayan RNAi levhalar vardır. En az 2 tabak, her durum için hazırlıklı olmalıdır. </li></ol> 2. RNAi Besleme Tabaklar Kültür Worms <ol><li> Alev bir platin tel solucan pick ucu sterilize. 6-10 dördüncü dönem larvaları (L4) ya rekombinant veya boş L4440 içeren her RNAi plakasına Hermafroditler almak almak solucan kullanın; hayvanların besin kaynağı olarak bakterileri kullanır. </li><li> Çift cinsiyetli 20 ° C (C. elegans için standart bir kültür koşulu) gecede büyümek edelim, onlar ertesi gün genç yetişkinler olacak. </li><li> Yeni buna etiketli ve hazırlanan RNAi plaka içine 6-10 genç yetişkinler aktarın. </li><li> Letyetişkinlerin 4-6 saat için yumurtalarını bıraktıkları, ardından plakası tüm yetişkinlerin kaldırmak; bu adımı döl senkronize etmektir. Anneler yok olduktan sonra, zaman saymaya başlar. Bu sefer sıfırdır. </li><li> 20 plakalar inkübe ° C hayvanların belirli bir gelişim evresine büyümesine izin vermek, bu protokolde, biz fenotipik analiz için genç yetişkinler seçin. Normal bir hızda gelişir hakemin için 72 saat inkübasyon genç yetişkinler haline hayvanlar için yeterlidir. Ancak, çeşitli genlerin farklı hayvan gelişimini etkileyebilir. RNAi farklı bir hızda büyümeye hayvanlar neden olursa, genç erişkinlerde rahim içinde tamamlanmış vulval geliştirme ve 2-6 embriyolar (fertil varsa) L4 geçmiş olanlar en fazla 24 saat olarak tespit edilebilir. Diğer gelişimsel aşamaları belirlemek için, araştırmacı bir rehber olarak gonadal ve vulval gelişimi kullanmalısınız. </li></ol> 3. RNAi Puan Vücut büyüklüğü Fenotiplerinin Worms Tedavi <ol><li> Bir cam slayt üzerine renkli etiket şeritleri iki kat yerleştirin. Of Make ikise cam slaytlar. Sonra, bant ile iki slayt arasına yeni bir cam slayt yerleştirin. Su içinde% 2 agaroz Melt yeni bir cam slayt merkezine bir damla uygulanır; sonra agaroz önceden 9 açıklanan% 2 ince bir katman yapmak için üstüne ikinci bir cam slayt basın. </li><li> Agaroz katılaşmış sonra, üst cam slayt kaldırmak; agaroz pad ile slayt solucanlar yüklemek için hazır. Klon adla Etiket slayt. </li><li> Agaroz pad 10 ul 25 mM NaN 3 ekleyin; onları hareketsiz NaN 3 solüsyon içine 30-40 hayvanları almak, sonra üstüne lamel koymak; rekombinant ve boş hem RNAi tedavi edilen hayvanlar L4440-taşıma için tekrar. </li><li> Görüntü 2.5x objektif lens kullanarak diseksiyon mikroskobu altında solucanlar; burada yazılım Qcapture destekleyen Leica dijital kamera kullanın. </li><li> Kalibrasyonu için, önce standart bir mikrometre cetvel bir görüntü yakalayabilirsiniz. Sonra Image-Pro yazılımı görüntüyü açmak seçin ve &quot;ölçü&quot; üzerine tıklayın ve &quot;kalibrasyon&quot; i seçin&quot;Mekansal kalibrasyon sihirbazı&quot;. Seçilen &quot;aktif görüntüyü kalibre&quot; ile &quot;gelecek&quot; üzerine tıklayın. Girdi kalibrasyon için isim ve mekansal referans birimleri mikrometre ayarlandığından emin olun. Sonra, &quot;oluşturmak için bir referans kalibrasyon&quot; düğmesini işaretleyin ve &quot;next&quot; butonuna tıklayınız. Tıklayın &quot;beraberlik referans çizgisi.&quot; Bir referans ölçek çubuğu görünecektir. Mikrometre ucuna uygun ölçek çubuğu yeniden konumlandırma, daha sonra 1000 adet referans gösterdiğini göstermektedir. , &quot;Tamam&quot;, &quot;Next&quot; ve &quot;Finish&quot; tıklayınız. </li><li> Yazılım kalibre edildikten sonra, bir solucan görüntüyü açmak. Sonra, &quot;select mekansal&quot; tıklayın sonra &quot;kalibrasyon&quot; seçin, &quot;tedbir&quot; menüsünün altında seçin. Açılan menüden ise, mikrometre kalibrasyonu için oluşturulan dosya adını seçin. Sonra, &quot;tedbir&quot; menüsü altında, &quot;ölçüm&quot; seçeneğini seçin. Açılan pencerede, serbest çizim aracını seçin ve bilgisayar faresi (Şekil 1) ile kuyruk baş hayvan vücudunun ortasından geçen bir çizgi izlemesi. Uzunluk pencere içinde rapor edilir. ŞimdiHedef gen RNAi L4440 ve boş vektör ile tedavi edilen kontrol hayvanları ile muamele edilen hayvanların vücut süresi: iki veri gruplarına sahiptir. </li><li> Microsoft Excel dosyası veya başka uygun istatistik yazılım uzunluk ölçümleri aktarın. Her numune için ortalama ve standart sapma hesaplanarak verileri analiz. Sonra Student t-testi kullanılarak örnekleri karşılaştırın. </li></ol>

<ol>
	<li>Melnyk, C. W., Molnar, A., Baulcombe, D. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intercellular+and+systemic+movement+of+RNA+silencing+signals.">Intercellular and systemic movement of RNA silencing signals.</a> Embo J. 30, 3553-3563 (2011).</li><li>Wall, N. R., Shi, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Small+RNA:+can+RNA+interference+be+exploited+for+therapy.">Small RNA: can RNA interference be exploited for therapy.</a> Lancet. 362, 1401-1403 (2003).</li><li>Kalantidis, K., Schumacher, H. T., Alexiadis, T., Helm, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNA+silencing+movement+in+plants.">RNA silencing movement in plants.</a> Biol. Cell. 100, 13-26 (2008).</li><li>Shim, M. S., Kwon, Y. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+and+targeted+delivery+of+siRNA+in+vivo.">Efficient and targeted delivery of siRNA in vivo.</a> Febs. J. 277, 4814-4827 (2010).</li><li>Amarzguioui, M., Rossi, J. J., Kim, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Approaches+for+chemically+synthesized+siRNA+and+vector-mediated+RNAi.">Approaches for chemically synthesized siRNA and vector-mediated RNAi.</a> FEBS Lett. 579, 5974-5981 (2005).</li><li>Kamath, R. S., Ahringer, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome-wide+RNAi+screening+in+Caenorhabditis+elegans.">Genome-wide RNAi screening in Caenorhabditis elegans.</a> Methods. 30, 313-321 (2003).</li><li>Savage-Dunn, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+screen+for+small+body+size+mutants+in+C.+elegans+reveals+many+TGFb+pathway+components.">Genetic screen for small body size mutants in C. elegans reveals many TGFb pathway components.</a> Genesis. 35, 239-247 (2003).</li><li>Timmons, L., Fire, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Specific+interference+by+ingested+dsRNA.">Specific interference by ingested dsRNA.</a> Nature. 395, 854 (1998).</li><li>Sulston, J. 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=Post-embryonic+cell+lineages+of+the+nematode,+Caenorhabditis+elegans.">Post-embryonic cell lineages of the nematode, Caenorhabditis elegans.</a> Dev. Biol. 56, 110-156 (1977).</li><li>Wang, J., Tokarz, R., Savage-Dunn, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+expression+of+TGF&#946;+signal+transducers+in+the+hypodermis+regulates+body+size+in+C.+elegans.">The expression of TGF&#946; signal transducers in the hypodermis regulates body size in C. elegans.</a> Development. 129, 4989-4998 (2002).</li><li>Liang, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Caenorhabditis+elegans+schnurri+homolog+sma-9+mediates+stage-+and+cell+type-specific+responses+to+DBL-1+BMP-related+signaling.">The Caenorhabditis elegans schnurri homolog sma-9 mediates stage- and cell type-specific responses to DBL-1 BMP-related signaling.</a> Development. 130, 6453-6464 (2003).</li><li>Savage-Dunn, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TGF-&#946;+signaling.">TGF-&#946; signaling.</a> WormBook. , 1-12 (2005).</li><li>Simmer, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Loss+of+the+putative+RNA-directed+RNA+polymerase+RRF-3+makes+C.+elegans+hypersensitive+to+RNAi.">Loss of the putative RNA-directed RNA polymerase RRF-3 makes C. elegans hypersensitive to RNAi.</a> Curr. Biol. 12, 1317-1319 (2002).</li><li>Wang, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Somatic+misexpression+of+germline+P+granules+and+enhanced+RNA+interference+in+retinoblastoma+pathway+mutants.">Somatic misexpression of germline P granules and enhanced RNA interference in retinoblastoma pathway mutants.</a> Nature. 436, 593-597 (2005).</li><li>De Robertis, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evo-devo:+variations+on+ancestral+themes.">Evo-devo: variations on ancestral themes.</a> Cell. 132, 185-195 (2008).</li><li>Timmons, L., Court, D. L., Fire, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ingestion+of+bacterially+expressed+dsRNAs+can+produce+specific+and+potent+genetic+interference+in+Caenorhabditis+elegans.">Ingestion of bacterially expressed dsRNAs can produce specific and potent genetic interference in Caenorhabditis elegans.</a> Gene. 263, 103-112 (2001).</li></ol>

Çıkar çatışması ilan etti.

Bu protokol, biz C. vücut büyüklüğü kusurlu mutantların belirlenmesi için yöntem tarif RNAi besleme ile elegans. Bu yöntem, TGF-β sinyal bileşenlerin tanımlanması için de geçerlidir. Gibi bileşenler yüksek evrim 15 yoluyla korunmuş olduğundan, bu ekranlar, tüm Metazoan&#39;da TGF-β sinyal moleküler mekanizmalarının aydınlatılmasına alakalı. Böyle bir ekran tasarımında önemli bir göz başlayarak suşudur. Lin-15b ve rrf-3 önceki çalışmal...

<table border="1"><tbody><tr><td></td><td></td><td></td><td> RNAi solucan plakaları 17.7 g Worm Orta Mix 60 g Agar 3 litre H 2 O ekleyin. Otoklavlayınız. 3 ml 1M IPTG (steril) ve 3 ml 25 mg / ml carbenicillin (steril) ekleyin ve sonra 60 mm Petri kapları içine dökün. Solucan plakaları 17.7 g Worm Orta Mix 0.6 g Streptomisin Sülfat 60 g Agar 3 litre H 2 O ekleyin. Otoklavlayınız. 60 mm Petri kapları içine dökün. Worm Orta Mix 55 g Tris-Cl, 24 g Tris-OH 310 g Bacto Peptonlu 800 mg Kolesterol 200 g NaCl İyice karıştırın ve topakları önlemek için emin olun, bu toz karışımı kullanmadan önce aylarca saklanabilir. % 2 agaroz 0.5 g Agaroz Iland 25 ml dH 2 O Mikrodalga Isı eriyene kadar. 1M izopropil β-D-1-thiogalactopyranoside (IPTG) 2 g IPTG In10 ml dH 2 O eritilir 1M NaN 3 6.5 g NaN3 100 ml dH 2 O ekleyin 25 mM NaN3 2.5 ml 1M NaN 3 100 ml dH 2 O ekleyin Caenorhabditis Genetik Merkezi&#39;nden Caenorhabditis elegans Plazmid L4440 (ampisilin direnç geni taşıyan) 16; Bakteri HT115 (DE3) (ayrıca tetrasiklin direnç geni taşıyan RNAse III geni (a dsRNAse) için eksik IPTG indüklenebilir T7 polimeraz içerir) zorlanma 60 mm Petri kapları 100 mm Petri kapları 14 ml&#39;lik yuvarlak tabanlı Falcon kültür tüpleri cam slaytlar cam lameller 1-20 ul pipet 20-200 ul pipet 200-1000 ulpipet 1-200 ul pipet ipuçları 200-1000 ul pipet ipuçları 1.5 ml Eppendorf tüpleri platin tel solucan almak </td></tr></tbody></table>

using rna-mediated interference feeding strategy to screen for genes involved in body size regulation in the nematode c. elegans

Çift iplikli RNA-aracılı girişim (RNAi) hedef gen ekspresyonu 1-3 yıkmak için etkili bir stratejidir. Bu bitkiler, omurgasızlar ve omurgalılar dahil birçok model sistemler uygulanmıştır. Vivo 4,5 RNAi ulaşmak için çeşitli yöntemler vardır. Örneğin, hedef gen, bir RNAi vektörü haline dönüştürülmesi ve daha sonra da sürekli veya geçici olarak hücre soyları ya da gen devirme etkileri elde etmek için birincil hücrelerine transforme edilebilir veya belirli bir hedef genin (RNAi Oligolar) için çift iplikli oligonükleotidler sentezlenebilir geçici olabilir hedef genleri susturmak için hücre hatları veya primer hücre dönüştürülmüştür; veya sentezlenen çift iplikli RNA molekülleri bir organizmanın içine mikroenjeksiyon yoluyla olabilir. Nematod C. yana elegans bakterileri hedef genlerin karşı çift iplikli RNA ifade ile hayvan besleme, bir gıda kaynağı olarak bakterileri kullanan uygun bir strateji 6. sağlar. Burada bir RNAi besleme sunmakyöntem vücut büyüklüğü fenotipi puan. C. Gövde boyutu elegans öncelikle TGF-β düzenlenir - gibi bir ligand DBL-1, böylece bu tahlil TGF-β sinyal bileşenleri 7 belirlenmesi için uygundur. Biz RNAi beslenme deneyleri tekrarlamak için iki RNAi duyarlı suşlar dahil farklı suşları kullanılmıştır. Bizim sonuçlarımız rrf-3 suşu bize en iyi beklenen RNAi fenotip verdiğini gösterdi. Yöntem, uygulanması kolay bir tekrarlanabilir ve kolayca ölçülür. Ayrıca, protokol özel ekipman kullanımını en aza indirir, bu nedenle daha küçük laboratuar ya da ağırlıklı olarak lisans kurumları da uygundur.

Araştırmamızda, biz DBL-1/TGF-β yolu ile vücut büyüklüğü düzenleme odaklanmak. Yabani tip hayvanlarda 7,10,11 ile karşılaştırıldığında daha kısa bir gövde uzunluk dahil olmak üzere küçük vücut boyutu DBL-1 yolunun sonuçları, fonksiyon kaybı. C. için Böylece, ekranlar elegans vücut büyüklüğü mutantlar TGF-β sinyal bileşenleri ve düzenleyici belirleme yeteneğine sahiptirler. DBL-1, sinyal hücre yüzey reseptörleri ve hücre içi sinyal dönüştürüc...

Watch this Scientific Journal Video about Using RNA-mediated Interference Feeding Strategy to Screen for Genes Involved in Body Size Regulation in the Nematode C. elegans at JoVE.com

Using RNA-mediated Interference Feeding Strategy to Screen for Genes Involved in Body Size Regulation in the Nematode C. elegans

Biz hedef genler ve skoru vücut büyüklüğüne fenotipi yıkmak için RNAi besleme tekniği nasıl kullanılacağını göstermektedir<em&gt; C. elegans</em&gt;. Bu yöntem, bu tür DBL-1/TGF-β sinyalleme yoluyla vücut boyutu düzenlenmesinde yer alan bu gibi muhtemel ilgi genetik bileşenler, belirlemek için büyük ölçekli bir ekran için kullanılabilir.

Nematod Vücut büyüklüğü Yönetmelik ilgilendim Genler için Ekran RNA-aracılı Girişim Besleme Strateji Kullanımı<em&gt; C. elegans</em

Double-strand RNA-mediated interference (RNAi) is an effective strategy to knock down target gene expression1-3. It has been applied to many model systems ...

using-rna-mediated-interference-feeding-strategy-to-screen-for-genes

Research

JoVE Journal

Biology

Using RNA-mediated Interference Feeding Strategy to Screen for Genes Involved in Body Size Regulation in the Nematode C. elegans

12.2K Görüntüleme.  Borough of Manhattan Community College, City Universtiy of New York (CUNY). Biz hedef genler ve skoru vücut büyüklüğüne fenotipi yıkmak için RNAi besleme tekniği nasıl kullanılacağını göstermektedir C. elegans. Bu yöntem, bu tür DBL-1/TGF-β sinyalleme yoluyla vücut boyutu düzenlenmesinde yer alan bu gibi muhtemel ilgi genetik bileşenler, belirlemek için büyük ölçekli bir ekran için kullanılabilir.

Video: Using RNA-mediated Interference Feeding Strategy to Screen for Genes Involved in Body Size Regulation in the Nematode C. elegans

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.

Application of a C. elegans Dopamine Neuron Degeneration Assay for the Validation of Potential Parkinson&#039;s Disease Genes

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 ...

Biyoloji

Isolation of Ribosome Bound Nascent Polypeptides in vitro to Identify Translational Pause Sites Along mRNA

The rate of translational elongation is non-uniform. mRNA secondary structure, codon usage and mRNA associated proteins may alter ribosome movement on the messagefor review see 1. However, it's now widely accepted that synonymous codon usage is the primary cause of non-uniform translational elongation rates1. Synonymous codons are not used with identical frequency. A bias exists in the use of synonymous codons with some codons used more frequently than others2. Codon bias is organism as well as tissue specific2,3. Moreover, frequency of codon usage is directly proportional to the concentrations of cognate tRNAs4. Thus, a frequently used codon will have higher multitude of corresponding tRNAs, which further implies that a frequent codon will be translated faster than an infrequent one. Thus, regions on mRNA enriched in rare codons (potential pause sites) will as a rule slow down ribosome movement on the message and cause accumulation of nascent peptides of the respective sizes5-8. These pause sites can have functional impact on the protein expression, mRNA stability and protein foldingfor review see 9. Indeed, it was shown that alleviation of such pause sites can alter ribosome movement on mRNA and subsequently may affect the efficiency of co-translational (in vivo) protein folding1,7,10,11. To understand the process of protein folding in vivo, in the cell, that is ultimately coupled to the process of protein synthesis it is essential to gain comprehensive insights into the impact of codon usage/tRNA content on the movement of ribosomes along mRNA during translational elongation.
Here we describe a simple technique that can be used to locate major translation pause sites for a given mRNA translated in various cell-free systems6-8. This procedure is based on isolation of nascent polypeptides accumulating on ribosomes during in vitro translation of a target mRNA. The rationale is that at low-frequency codons, the increase in the residence time of the ribosomes results in increased amounts of nascent peptides of the corresponding sizes. In vitro transcribed mRNA is used for in vitro translational reactions in the presence of radioactively labeled amino acids to allow the detection of the nascent chains. In order to isolate ribosome bound nascent polypeptide complexes the translation reaction is layered on top of 30% glycerol solution followed by centrifugation. Nascent polypeptides in polysomal pellet are further treated with ribonuclease A and resolved by SDS PAGE. This technique can be potentially used for any protein and allows analysis of ribosome movement along mRNA and the detection of the major pause sites. Additionally, this protocol can be adapted to study factors and conditions that can alter ribosome movement and thus potentially can also alter the function/conformation of the protein.

Isolation of Ribosome Bound Nascent Polypeptides in vitro to Identify Translational Pause Sites Along mRNA

A technique to identify translational pause sites on mRNA is described. This procedure is based on isolation of nascent polypeptides accumulating on ribosomes during in vitro translation of a target mRNA, followed by the size analysis of the nascent chains using a denaturing gel electrophoresis.

Ribozom Bağlı Doğan Polipeptitler İzolasyonu<em&gt; In vitro</em&gt; MRNA boyunca Translasyonel Duraklat Siteleri Belirleme

The rate of translational elongation is non-uniform. mRNA secondary structure, codon usage and mRNA associated proteins may alter ribosome movement on the ...

Using SecM Arrest Sequence as a Tool to Isolate Ribosome Bound Polypeptides

Extensive research has provided ample evidences suggesting that protein folding in the cell is a co-translational process1-5. However, the exact pathway that polypeptide chain follows during co-translational folding to achieve its functional form is still an enigma. In order to understand this process and to determine the exact conformation of the co-translational folding intermediates, it is essential to develop techniques that allow the isolation of RNCs carrying nascent chains of predetermined sizes to allow their further structural analysis.
SecM (secretion monitor) is a 170 amino acid E. coli protein that regulates expression of the downstream SecA (secretion driving) ATPase in the secM-secA operon6. Nakatogawa and Ito originally found that a 17 amino acid long sequence (150-FSTPVWISQAQGIRAGP-166) in the C-terminal region of the SecM protein is sufficient and necessary to cause stalling of SecM elongation at Gly165, thereby producing peptidyl-glycyl-tRNA stably bound to the ribosomal P-site7-9. More importantly, it was found that this 17 amino acid long sequence can be fused to the C-terminus of virtually any full-length and/or truncated protein thus allowing the production of RNCs carrying nascent chains of predetermined sizes7. Thus, when fused or inserted into the target protein, SecM stalling sequence produces arrest of the polypeptide chain elongation and generates stable RNCs both in vivo in E. coli cells and in vitro in a cell-free system. Sucrose gradient centrifugation is further utilized to isolate RNCs.
The isolated RNCs can be used to analyze structural and functional features of the co-translational folding intermediates. Recently, this technique has been successfully used to gain insights into the structure of several ribosome bound nascent chains10,11. Here we describe the isolation of bovine Gamma-B Crystallin RNCs fused to SecM and generated in an in vitro translation system.

We describe here a technique that is now routinely used to isolate stably bound ribosome nascent chain complexes (RNCs). This technique takes advantage of the discovery that a 17 amino acid long SecM "arrest sequence" can halt translation elongation in a prokaryotic (E. coli) system, when inserted into (or fused to the C-terminus) of virtually any protein.

Ribozom Ciltli Polipeptitler izole bir aracı olarak SECM Tutuklanma sırası kullanılarak

Extensive research has provided ample evidences suggesting that protein folding in the cell is a co-translational process1-5. However, the exact pathway ...

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 &quot;Freeze-Cracking&quot;

"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 ...

Large-scale Gene Knockdown in C. elegans Using dsRNA Feeding Libraries to Generate Robust Loss-of-function Phenotypes

RNA interference by feeding worms bacteria expressing dsRNAs has been a useful tool to assess gene function in C. elegans. While this strategy works well when a small number of genes are targeted for knockdown, large scale feeding screens show variable knockdown efficiencies, which limits their utility. We have deconstructed previously published RNAi knockdown protocols and found that the primary source of the reduced knockdown can be attributed to the loss of dsRNA-encoding plasmids from the bacteria fed to the animals. Based on these observations, we have developed a dsRNA feeding protocol that greatly reduces or eliminates plasmid loss to achieve efficient, high throughput knockdown. We demonstrate that this protocol will produce robust, reproducible knock down of C. elegans genes in multiple tissue types, including neurons, and will permit efficient knockdown in large scale screens. This protocol uses a commercially available dsRNA feeding library and describes all steps needed to duplicate the library and perform dsRNA screens. The protocol does not require the use of any sophisticated equipment, and can therefore be performed by any C. elegans lab.

Large-scale Gene Knockdown in C. elegans Using dsRNA Feeding Libraries to Generate Robust Loss-of-function Phenotypes

While dsRNA feeding in C. elegans is a powerful tool to assess gene function, current protocols for large scale feeding screens result in variable knockdown efficiencies. We describe an improved protocol for performing large scale RNAi feeding screens that results in highly efficacious and reproducible knockdown of gene expression.

Büyük ölçekli Gen demonte olarak<em&gt; C. elegans</em&gt; Sağlam Kayıp fonksiyon-fenotipleri oluşturmak için DsRNA Besleme Kitaplıklar kullanma

RNA interference by feeding worms bacteria expressing dsRNAs has been a useful tool to assess gene function in C. elegans. While this strategy works well ...

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

The folding and assembly of proteins is essential for protein function, the long-term health of the cell, and longevity of the organism. Historically, the function and regulation of protein folding was studied in vitro, in isolated tissue culture cells and in unicellular organisms. Recent studies have uncovered links between protein homeostasis (proteostasis), metabolism, development, aging, and temperature-sensing. These findings have led to the development of new tools for monitoring protein folding in the model metazoan organism Caenorhabditis elegans. In our laboratory, we combine behavioral assays, imaging and biochemical approaches using temperature-sensitive or naturally occurring metastable proteins as sensors of the folding environment to monitor protein misfolding. Behavioral assays that are associated with the misfolding of a specific protein provide a simple and powerful readout for protein folding, allowing for the fast screening of genes and conditions that modulate folding. Likewise, such misfolding can be associated with protein mislocalization in the cell. Monitoring protein localization can, therefore, highlight changes in cellular folding capacity occurring in different tissues, at various stages of development and in the face of changing conditions. Finally, using biochemical tools ex vivo, we can directly monitor protein stability and conformation. Thus, by combining behavioral assays, imaging and biochemical techniques, we are able to monitor protein misfolding at the resolution of the organism, the cell, and the protein, respectively.

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

To study the relationship between protein homeostasis, stress and aging, we monitored changes in protein folding by following protein dysfunction, protein localization in the cell and protein stability at the organismal, cellular and protein levels, using the genetically tractable metazoan Caenorhabditis elegans as a model system.

The folding and assembly of proteins is essential for protein function, the long-term health of the cell, and longevity of the organism. Historically, the ...

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 ...

Tracking and Quantifying Developmental Processes in C. elegans Using Open-source Tools

Quantitatively capturing developmental processes is crucial to derive mechanistic models and key to identify and describe mutant phenotypes. Here protocols are presented for preparing embryos and adult C. elegans animals for short- and long-term time-lapse microscopy and methods for tracking and quantification of developmental processes. The methods presented are all based on C. elegans strains available from the Caenorhabditis Genetics Center and on open-source software that can be easily implemented in any laboratory independently of the microscopy system used. A reconstruction of a 3D cell-shape model using the modelling software IMOD, manual tracking of fluorescently-labeled subcellular structures using the multi-purpose image analysis program Endrov, and an analysis of cortical contractile flow using PIVlab (Time-Resolved Digital Particle Image Velocimetry Tool for MATLAB) are shown. It is discussed how these methods can also be deployed to quantitatively capture other developmental processes in different models, e.g., cell tracking and lineage tracing, tracking of vesicle flow.

Tracking and Quantifying Developmental Processes in C. elegans Using Open-source Tools

Here it is shown how to track and quantify developmental processes in C. elegans. The methods presented are based on open-source tools that can be easily implemented. It is demonstrated how to reconstruct 3D cell-shape models, how to manually track subcellular structures, and how to analyze cortical contractile flow.

Gelişim Süreçleri İzleme ve nicel<em&gt; C. elegans</em&gt; Açık kaynak Araçlarını Kullanma

Quantitatively capturing developmental processes is crucial to derive mechanistic models and key to identify and describe mutant phenotypes. Here ...

Measuring RAN Peptide Toxicity in C. elegans

C. elegans is commonly used to model age-related neurodegenerative diseases caused by repeat expansion mutations, such as Amyotrophic Lateral Sclerosis (ALS) and Huntington&#8217;s disease. Recently, repeat expansion-containing RNA was shown to be the substrate for a novel type of protein translation called repeat-associated non-AUG-dependent (RAN) translation. Unlike canonical translation, RAN translation does not require a start codon and only occurs when repeats exceed a threshold length. Because there is no start codon to determine the reading frame, RAN translation occurs in all reading frames from both sense and antisense RNA templates that contain a repeat expansion sequence. Therefore, RAN translation expands the number of possible disease-associated toxic peptides from one to six. Thus far, RAN translation has been documented in eight different repeat expansion-based neurodegenerative and neuromuscular diseases. In each case, deciphering which RAN products are toxic, as well as their mechanisms of toxicity, is a critical step towards understanding how these peptides contribute to disease pathophysiology. In this paper, we present strategies to measure the toxicity of RAN peptides in the model system C. elegans. First, we describe procedures for measuring RAN peptide toxicity on the growth and motility of developing C. elegans. Second, we detail an assay for measuring postdevelopmental, age-dependent effects of RAN peptides on motility. Finally, we describe a neurotoxicity assay for evaluating the effects of RAN peptides on neuron morphology. These assays provide a broad assessment of RAN peptide toxicity and may be useful for performing large-scale genetic or small molecule screens to identify disease mechanisms or therapies.

Measuring RAN Peptide Toxicity in C. elegans

Repeat-associated non-ATG-dependent translational products are emerging pathogenic features of several repeat expansion-based diseases. The goal of the protocol described is to evaluate toxicity caused by these peptides using behavioral and cellular assays in the model system C. elegans.

C. elegans RAN Peptid Toksisitesi Ölçme

C. elegans is commonly used to model age-related neurodegenerative diseases caused by repeat expansion mutations, such as Amyotrophic Lateral Sclerosis ...

Using Caenorhabditis elegans to Screen for Tissue-Specific Chaperone Interactions

Correct folding and assembly of proteins and protein complexes are essential for cellular function. Cells employ quality control pathways that correct, sequester or eliminate damaged proteins to maintain a healthy proteome, thus ensuring cellular proteostasis and preventing further protein damage. Because of redundant functions within the proteostasis network, screening for detectable phenotypes using knockdown or mutations in chaperone-encoding genes in the multicellular organism Caenorhabditis elegans results in the detection of minor or no phenotypes in most cases. We have developed a targeted screening strategy to identify chaperones required for a specific function and thus bridge the gap between phenotype and function. Specifically, we monitor novel chaperone interactions using RNAi synthetic interaction screens, knocking-down chaperone expression, one chaperone at a time, in animals carrying a mutation in a chaperone-encoding gene or over-expressing a chaperone of interest. By disrupting two chaperones that individually present no gross phenotype, we can identify chaperones that aggravate or expose a specific phenotype when both perturbed. We demonstrate that this approach can identify specific sets of chaperones that function together to modulate the folding of a protein or protein complexes associated with a given phenotype.

Using Caenorhabditis elegans to Screen for Tissue-Specific Chaperone Interactions

To study chaperone-chaperone and chaperone-substrate interactions, we perform synthetic interaction screens in Caenorhabditis elegans using RNA interference in combination with mild mutations or over-expression of chaperones and monitor tissue-specific protein dysfunction at the organismal level.

Dokuya Özgü Chaperone Etkileşimlerini Taramak için Caenorhabditis elegans'ı Kullanma

Correct folding and assembly of proteins and protein complexes are essential for cellular function. Cells employ quality control pathways that correct, ...