C. elegans transgenlerin Caenorhabditis Genetik Merkezi (Minnesota Üniversitesi, Minneapolis, MN) tarafından verilmiştir. Bu çalışma NIH R21 hibe NS0667678 KS-01 tarafından desteklenmiştir. Tüm yazarlar katıldı toplanması, analizi ve verilerin yorumlanması. CKL, KS ve KMG yazının yazımı ve gözden geçirilmesi katıldı.

1. Bakteriyel solucan gıda hazırlanması 1. Gün <ol><li> 5 ml doymuş E. ekle 50 mg / ml streptomisin coli OP50 bakteri kültürü 500 ml Terrific suyu ile desteklenmiş ve sallayarak inkübatör (225 rpm) bir gecede büyümek 37 ° C </li></ol> 2. Gün <ol start="2"><li> 20 dakika boyunca 2500 RCF soğutmalı santrifüj on 50 ml tüpler ve santrifüj bakteri içine Split gecede bakteri kültürü. </li><li> LB suyu kapalı Durusu ve 10 ml sıvı nematod büyüme ortam (NGM) her bakteriyel pelet tekrar süspansiyon haline getirin. Bakterilerin tekrar süspansiyon 15 dakika boyunca yatay bir zemin çalkalayıcı çalkalayınız. NGM tampon yapmak için 3 gr NaCl, 1 L deiyonize su ve otoklav ekleyin. Serin - 55 ° C ve steril çözümler için aşağıdaki ekleyin: 1 ml 1 M CaCl 2, 1 M MgSO 4 1 ml, 25 ml ve 1 M potasyum fosfat, pH 6.0 . </li><li> 20 dakika boyunca 2500 RCF soğutmalı santrifüj bakteri kültürü santrifüjleyin. </li><li> NGM tampon kapalı Durusu ve bakteriyel pelet tartmak. </li><li> Granül, kısım bakteri 3 ml, 15 ml tüpler ve mağaza içine konsantre -20 ° C tekrar süspansiyon NGM tampon eşit hacimde </li></ol> 2. C. Büyük ölçekli elegans sıvı kültürü Biz bir transgenik satırını kullanın VP596 (dvIs19 [pAF15 (gst-4:: GFP: NLS)]; vsIs33 [dop-3: TÇD) iki floresan yapıları taşıyan SKN-1 izlemek için GFP 14: Pgst: -4 aktivite ve Pdop-3: RFP 17 solucan sayısı normalleşmesi için bir standart olarak hizmet vermektedir. 1. Gün <ol><li> 150 ml, 1.5 ml LB suyu, 5 mg / ml &#39;kolesterol (etanol) 150 ul, 100 mg / ml streptomisin 75 ul NGM tampon karıştırın. Karışımı Filtre sterilize ve 1 litre steril erlene ekleyin. NOT:% 1 LB suyu NGM tampon ekleme, solucanlar, cam ve plasticware yapışmasını önlemek için yardımcı olur, ancak bu miktar bakteri büyümeyi desteklemek için yeterli değildir. </li><li> Solucanlar standart hipoklorit prosedürü 18 ile senkronize etme. Hipoklorit solüsyonu 5 ml (3.75 ml steril su, 1 ml çamaşır suyu, ve 250 ul 10 N NaOH) ile 0.5 ml gravid solucanlar, tek bir 15 ml tüp içinde işlenebilir. Bırakılan yumurta steril su ile yıkayın ve 10 ml NGM tampon tekrar süspansiyon haline getirin. </li><li> Yumurta NGM tampon 100 kat (örneğin, 10 ml 100 ul) bir örnek seyreltilir ve üç ayrı 5 ul alikotları yumurta sayısını. Çoklu 200.000 (100 seyreltme faktörü x 2,000), ortalama toplam yumurta sayısını tahmin etmek. </li><li> NGM tamponu ile şişesi için 200.000 &#39;e 2 milyon yumurta ekleyin ve kültür sallamak 100 rpm&#39;de 20 ° C. </li></ol> 2. Gün <ol start="5"><li> Erlene yumurta ekledikten sonra en az 16 saat asılı bir solucan kültür yaklaşık 0,5 ml kaldırmak için steril bir serolojik pipetlemeyin kullanın. Petri kabı steril kapak üzerine üç pipetleyin 5 ul damla. Solucanlar felç için 1-2 dakika boyunca -20 ° C derin dondurucu kapağı yerleştirin. Yumurtadan solucanlar toplam sayısını tahmin etmek için ortalama sayısı 30.000 (150 ml / 5 ul) 5 ul ve sonra birden başına canlı yumurtadan solucanlar güvenin. </li><li> Dondurulmuş OP50 bakteri kültürü (Protokol 1.6) bir tüp çözülme ve 500.000 yumurtadan solucanlar başına solucan kültürü içine 3 ml% 50 OP50 ekleyin. Şişeyi çalkalayın 100 rpm&#39;de 20 ° C. Worms L4 larva ve genç erişkinlerde yaklaşık 51 saat içinde dönüşecek. Bakteri OD600 başında 2.200 hakkında olmalıdır. Absorbans bakteri miktarı Monitör ve daha eklemek OD 600 aşağıdaki 0.900 düşerse. </li></ol> 3. Solucan toplama ve dağıtım 4. Gün <ol><li> Standart bir NGM agar plaka askıya solucan kültür yaklaşık 0.5 ml transfer etmek için steril bir serolojik pipetlemeyin kullanın. En L4 larva veya genç erişkinlerde olduğunu emin olmak için bir stereomikroskopta solucanlar. L4 larvaları ventral vücut ortasında bir açık nokta olacaktır. Genç yetişkinler, biraz daha büyük ve net bir nokta olmayacaktır olacak. </li><li> Solucan kültürü steril 50 ml tüpler içine dökün ve bir test tüpü rakı benchtop tüpler. Solucanlar, yaklaşık 10 dakika boyunca yerleşmek için izin verin. Aspirasyonu veya pipetleme süpernatantı. NOT: Bu adım, L4 larva ve genç erişkinlerde daha yavaş yerleşmeye çünkü çıkmamış yumurta veya geliştirmek değil solucanları kaldırmak için önemli olabilir. </li><li> NGM tamponu ile 1% LB suyu (NGM + LB) bakteriler kaldırmak için 3-4 kez 50 ml tek bir tüp ve yıkama tüm solucanları toplayın. NOT: Bu en iyi 30 saniye için 500 RCF solucanlar toplama ve taze NGM + LB tampon ile süpernatantı yerine bir sallanan kovası santrifüj yapılır. </li><li> Yıkadıktan sonra, 50 ml + NGM LB tampon tüp doldurmak ve özel bir solucan dağıtımı şişesi içine dökünüz. Küçük bir heyecan çubuğuna ekleyin ve solucanlar askıda tutmak için karıştırın. Kasım Sayısıüç 5 ul damla solucanlar e numarası yukarıda da belirtildiği gibi. 5 ul damla (~ 2,5-3,0 solucanlar / ml) ortalama 12-15 solucanlar kadar NGM + LB ile sulandırınız. NOT: yaklaşık 20 ml (60.000 solucanlar) en az dağıtım şişesi ve dağıtıcı kaset ölü boşluğu doldurmak için gereklidir. Her 384 plaka yaklaşık 35.000 solucan veya askıya solucanlar 11.5 ml gerektirir. </li><li> 10 ul dağıtım kaset Yük ve% 70 etanol ile astarlanırsa ile sterilize edin. Steril su ile astarlanırsa etanol durulayın. </li><li> Sadece hareketini bozmadan heyecan bar üstünde olduğunu balona dağıtım kaset takın. Solucanlar tüm şişesi boyunca askıda kalır emin olun. Program, dağıtıcı, 2 pre-bakliyat ile düşük hızda dağıtmak için. Başbakan ve askıya solucanlar en az 10 ml çalıştırın. Plakalar, tüp yerleşen solucanları önlemek için hızlı bir şekilde gerektiği gibi doldurun. </li><li> Nefes bant ile plakaları. </li><li> Tayini için uygun sıcaklıkta bir inkübatör sallayarak platformu tabak koyun. Plakalar koymayın. </li></ol> 4. Floresans analizi <ol><li> Uygun emisyon ve dalgaboyu (:; RFP 540/25ex 590/35em GFP 485/20ex 528/20em tayini için Filtreler) ile her bir kuyunun floresan yoğunluğu ölçmek için bir mikroplaka okuyucu hedef plakası yerleştirin. </li><li> GFP / RFP oranını hesaplayın ve bireysel tedavi edilen floresan yoğunluğu tam kat farkı belirlemek için kontrol kuyuları (aktive bileşik olmadan) okuma normalleştirmek. </li></ol> 5. Temsilcisi Sonuçlar: Bizim SKN-1 testinin kromozomal olarak entegre bir çift muhabiri suşu (VP596) kullanır. Şekil 1&#39;de gösterildiği gibi, solucanlar sayıda reçete hacmi de ilişkilidir. De ortalama Şekil 2A ve 2B, toplam GFP ve RFP floresan görüldüğü gibi son derece iyi bir 384 plaka iyi çapında tekrarlanabilir. RFP çapında 384 kuyu: GFP floresan doğrusal Pdop-3 ile korele: un bağlı Pgst-4 Şekil 2C, gösterilmiştir. Değişkenliği azaltmak için RFP muhabiri: GFP / RFP bir oranı olarak ifade edildiğinde, floresan Pdop-3 yeteneğini gösteren 384 plaka (2B Şekil 2A varyasyon katsayısı karşılaştırmak) karşısında kuyudan yüksek tekrarlanabilir hale gelir . Şekil 3 ile RFP GFP göreli indüksiyon gösterildiği gibi bir SKN-1 ksenobiyotik aktive (38 mcM juglone&#39;un) 384 plaka boyunca son derece sağlam ve tekrarlanabilir. <img src="/files/ftp_upload/2745/2745fig1.jpg" alt="Şekil 1"/> Şekil 1 hacim karşı solucanlar sayısı vazgeçtik. Worms yaklaşık 2/μl seyreltilir ve bir stereomikroskopta manuel sayım için 24 plaka halinde dağıtıldı. N = 8 kuyu hacim başına. <img src="/files/ftp_upload/2745/2745fig2.jpg" alt="Şekil 2"/> Şekil 2. Toplam 384 plaka boşaltılan solucanlar floresan tekrarlanabilir. Worms, yaklaşık 2.5/μl ve 30 ul seyreltilir 384 plaka her kuyuya dağıtılır oldu. Tüm kuyuların GFP (A) ve TTT (B) floresan varyasyon katsayısı% 9 altında vardı. (C) GFP floresan yüksek RFP floresan ilişkilidir. (D)% 6 altında RFP için GFP oranı hesaplanması için varyasyon katsayısı azaltılmış (A, B, ve D) Düz çizgiler anlamına gelir gösterir ve kırık çizgiler, ortalamanın altında veya üstünde üç standart sapma gösterir. <img src="/files/ftp_upload/2745/2745fig3.jpg" alt="Şekil 3"/> Pgst-4 Şekil 3 Etkinleştirme: GFP sağlam ve tutarlı. Yaklaşık 75 L4 solucanlar 384 plaka ve 38 mcM juglone&#39;un tüm kuyulara dağıtıldı diğer her sütun eklendi. GFP ve RFP floresan inkübasyon 21 saat sonra ölçüldü. Ortalama bağıl floresan tüm kontrol oranları (1.0) ve juglone&#39;un kuyuları (8,9) katı çizgilerle işaretlenmiştir. Ortalama kontrol yukarıdaki üç standart sapma ve juglone&#39;un ortalama altında kırık hatları ile işaretlenmiştir.

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Biz kültür için bir yöntem mevcut ve transgenik nematodların çok sayıda dağıtım. Kültür solucanları için kullanılan ekipman, moleküler klonlama gerçekleştiren laboratuarlar için standart ve sıvı taşıma ve çok sayıda mikropleytin işleme laboratuarları için standart floresan ekipman. Diğer çok sayıda canlı C. dağıtım yöntemleri elegans pahalı ekipmanlar 19 sıralama parçacık gerektirir. Pgst-4: GFP tahlil kap &#39;n&#39; yaka transkripsiyon fakt?...

<table border="1"><tbody><tr><th> Reaktifin Adı </th><th> Şirket </th><th> Katalog numarası </th><th> Yorumlar (isteğe bağlı) </th></tr><tr><td> LB besiyeri </td><td> Araştırma Ürünler International Corp </td><td> L24066 </td><td></td></tr><tr><td> Terrific suyu </td><td> Araştırma Ürünler International Corp </td><td> T15100 </td><td></td></tr><tr><td> Synergy ™ HT Multi-mode Mikroplaka Okuyucu </td><td> BioTek </td><td></td><td> Filtreler: GFP 485/20ex 528/20em; RFP 540/25ex 590/35em </td></tr><tr><td> MicroFlo seçin Dispenseri </td><td> BioTek </td><td></td><td></td></tr><tr><td> Solucan dağıtım şişesi </td><td> Güney Scientific Inc, Micanopy, FL </td><td></td><td> Özel (352) 284-2531 toplandı </td></tr><tr><td> 384 mikroplaka </td><td> Greiner Bio-One </td><td> 5678-1209 </td><td></td></tr><tr><td> Nefes sızdırmazlık bandı </td><td> Nunc </td><td> 241205 </td><td></td></tr><tr><td> (5-hidroksi-p-naphthoqinone) juglone&#39;un </td><td> ACROS </td><td> 121640010 </td><td> Juglone&#39;un DMSO içinde çözülür </td></tr><tr><td> DMSO </td><td> Sigma-Aldrich </td><td> D-5879 </td><td></td></tr><tr><td> C. elegans transgenik suşu </td><td> Yazarın laboratuvarı </td><td> VP596 </td><td> Pgst-4: GFP ve Pdop-3: RFP </td></tr></tbody></table>

high-throughput screening and biosensing with fluorescent c. elegans strains

Yüksek verim tarama (HTS), biyolojik süreçler kimyasal modülatörleri tanımlamak için güçlü bir yaklaşımdır. Ancak, hücre kültürü modelleri kullanarak ekranlar tanımlanan birçok bileşikler genellikle, in vivo 1-2 toksik ya da farmakolojik olarak inaktif olarak bulunmuştur . Tüm hayvan modellerinde de Eleme Bu tuzaklardan kaçınmak ve ilaç geliştirme yolunu düzene yardımcı olabilir. C. elegans, iyi HTS için uygun bir çok hücreli bir model organizmadır. Küçük (&lt;1 mm) ve ekonomik kültürlü ve sıvılarda reçete edilmesi C. elegans, ilaç modu-aksiyon 3 hızlı ve ayrıntılı kimlik izin, en deneysel olarak izlenebilir hayvan modellerinde de biridir. Biz kültür için bir protokol tanımlayan ve C floresan suşları dağıtım kimyasal kütüphaneleri veya belirli bir gen ifadesini değiştiren çevresel kirleticiler tespit yüksek verim tarama için elegans. Çok sayıda gelişimsel senkronize solucanlar, sıvı kültüründe yetişen hasat, yıkanmış ve belirli bir yoğunlukta asılı. Worms peristaltik bir sıvı dağıtıcı kullanarak siyah, düz tabanlı 384-kuyucuğu sonra eklenir. Küçük moleküllerin kimyasal bir kütüphane ya da test örnekleri (örneğin, su, gıda, veya toprak), solucanlar ile kuyu eklenebilir. In vivo olarak, gerçek zamanlı floresan bir floresan mikroplak okuyucu ile ölçülür . Bu yöntem, C herhangi bir indüklenebilir gen adapte edilebilir uygun bir muhabir kullanılabilir olduğu elegans. Birçok indüklenebilir stres ve gelişim transkripsiyonel yollar C ile tanımlanır elegans ve GFP transgenik muhabiri suşları zaten onları 4 pek çok var. Uygun transgenik gazetecilere ile kombine edildiğinde, yöntem yolağı modülatörleri ekrana veya çevresel kirleticiler için sağlam bir biyosensör deneyleri geliştirmek için kullanılabilir. Biz C. göstermektedir elegans kültür ve dağıtım protokolü bir HTS testi ile C. izlemek için geliştirilen elegans kap &#39;n&#39; yaka transkripsiyon faktörü SKN-1. SKN-1 ve memeli homologu Nrf2 5-10 ve ksenobiyotik oksidatif stres sırasında sitoprotektif genler aktif hale getirin. Nrf2 memeliler, kanser nörodejenerasyon ve kronik inflamasyon gibi pek çok yaşa bağlı bozukluklar korur ve büyük bir kemoterapötik hedef 11-13 haline gelmiştir. Tahlil -4 SKN-1 hedef geni gst 14 GFP transgenik muhabiri dayalı , glutatyon-s transferaz 6 kodlar. Gst -4 muhabiri SKN-1 etkinleştirin ve akrilamid ve metil-civa 15-16 gibi kirleticilerin düşük seviyelerde tespit etmek için kullanılan olabilir ksenobiyotik ve oksidatif kimyasallar için de bir biyosensör.

Watch this Scientific Journal Video about High-throughput Screening and Biosensing with Fluorescent C. elegans Strains at JoVE.com

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

Sıvı tabanlı kültür ve dağıtımı için bir prosedür<em&gt; C. elegans</emFloresan muhabiri proteinleri ifade&gt; suşları pahalı sıralama ekipman gerektirmez açıklanmıştır. Bu yaklaşım çok sayıda indüklenebilir uygulanabilir<em&gt; C. elegans</em&gt; Ilaç keşfi veya bulaşanların biyoalgı için genlerin.

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 ...

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Research

JoVE Journal

Biology

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

17.8K Görüntüleme.  University of Florida. Sıvı tabanlı kültür ve dağıtımı için bir prosedür C. elegans suşları pahalı sıralama ekipman gerektirmez açıklanmıştır. Bu yaklaşım çok sayıda indüklenebilir uygulanabilir C. elegans Ilaç keşfi veya bulaşanların biyoalgı için genlerin.

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

High Throughput Screening of Fungal Endoglucanase Activity in Escherichia coli

Cellulase enzymes (endoglucanases, cellobiohydrolases, and &beta;-glucosidases) hydrolyze cellulose into component sugars, which in turn can be converted into fuel alcohols1. The potential for enzymatic hydrolysis of cellulosic biomass to provide renewable energy has intensified efforts to engineer cellulases for economical fuel production2. Of particular interest are fungal cellulases3-8, which are already being used industrially for foods and textiles processing.
Identifying active variants among a library of mutant cellulases is critical to the engineering process; active mutants can be further tested for improved properties and/or subjected to additional mutagenesis. Efficient engineering of fungal cellulases has been hampered by a lack of genetic tools for native organisms and by difficulties in expressing the enzymes in heterologous hosts. Recently, Morikawa and coworkers developed a method for expressing in E. coli the catalytic domains of endoglucanases from H. jecorina3,9, an important industrial fungus with the capacity to secrete cellulases in large quantities. Functional E. coli expression has also been reported for cellulases from other fungi, including Macrophomina phaseolina10 and Phanerochaete chrysosporium11-12. 
We present a method for high throughput screening of fungal endoglucanase activity in E. coli. (Fig 1) This method uses the common microbial dye Congo Red (CR) to visualize enzymatic degradation of carboxymethyl cellulose (CMC) by cells growing on solid medium. The activity assay requires inexpensive reagents, minimal manipulation, and gives unambiguous results as zones of degradation (&#x201C;halos&#x201D;) at the colony site. Although a quantitative measure of enzymatic activity cannot be determined by this method, we have found that halo size correlates with total enzymatic activity in the cell. Further characterization of individual positive clones will determine , relative protein fitness. 
Traditional bacterial whole cell CMC/CR activity assays13 involve pouring agar containing CMC onto colonies, which is subject to cross-contamination, or incubating cultures in CMC agar wells, which is less amenable to large-scale experimentation. Here we report an improved protocol that modifies existing wash methods14 for cellulase activity: cells grown on CMC agar plates are removed prior to CR staining. Our protocol significantly reduces cross-contamination and is highly scalable, allowing the rapid screening of thousands of clones. In addition to H. jecorina enzymes, we have expressed and screened endoglucanase variants from the Thermoascus aurantiacus and Penicillium decumbens (shown in Figure 2), suggesting that this protocol is applicable to enzymes from a range of organisms.

High Throughput Screening of Fungal Endoglucanase Activity in Escherichia coli

We describe a low cost, high throughput method to screen for fungal endoglucanase activity in E. coli. The method relies on a simple visual readout of substrate degradation, does not require enzyme purification, and is highly scalable. This allows for the rapid screening of large libraries of enzyme variants.

Mantar Endoglucanase Faaliyet Yüksek Verimli Tarama Escherichia coli</em

Cellulase enzymes (endoglucanases, cellobiohydrolases, and &beta;-glucosidases) hydrolyze cellulose into component sugars, which in turn can be converted ...

Biyoloji

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 and Automated High-throughput Genome-wide RNAi Screens in C. elegans

RNA interference is a powerful method to understand gene function, especially when conducted at a whole-genome scale and in a quantitative context. In C. elegans, gene function can be knocked down simply and efficiently by feeding worms with bacteria expressing a dsRNA corresponding to a specific gene 1. While the creation of libraries of RNAi clones covering most of the C. elegans genome 2,3 opened the way for true functional genomic studies (see for example 4-7), most established methods are laborious. Moy and colleagues have developed semi-automated protocols that facilitate genome-wide screens 8. The approach relies on microscopic imaging and image analysis. 
	Here we describe an alternative protocol for a high-throughput genome-wide screen, based on robotic handling of bacterial RNAi clones, quantitative analysis using the COPAS Biosort (Union Biometrica (UBI)), and an integrated software: the MBioLIMS (Laboratory Information Management System from Modul-Bio) a technology that provides increased throughput for data management and sample tracking. The method allows screens to be conducted on solid medium plates. This is particularly important for some studies, such as those addressing host-pathogen interactions in C. elegans, since certain microbes do not efficiently infect worms in liquid culture.
	We show how the method can be used to quantify the importance of genes in anti-fungal innate immunity in C. elegans. In this case, the approach relies on the use of a transgenic strain carrying an epidermal infection-inducible fluorescent reporter gene, with GFP under the control of the promoter of the antimicrobial peptide gene nlp 29 and a red fluorescent reporter that is expressed constitutively in the epidermis. The latter provides an internal control for the functional integrity of the epidermis and nonspecific transgene silencing9. When control worms are infected by the fungus they fluoresce green. Knocking down by RNAi a gene required for nlp 29 expression results in diminished fluorescence after infection. Currently, this protocol allows more than 3,000 RNAi clones to be tested and analyzed per week, opening the possibility of screening the entire genome in less than 2 months.

Quantitative and Automated High-throughput Genome-wide RNAi Screens in C. elegans

We describe a protocol using C. elegans and RNAi feeding libraries that allows automated measurement of multiple parameters such as fluorescence, size and opacity of individual worms in a population. We give one example of a screen to identify genes involved in anti-fungal innate immunity in C. elegans.

Nicel ve Otomatik Yüksek throughput Genom çapında RNAi Ekranlar C. elegans</em

RNA interference is a powerful method to understand gene function, especially when conducted at a whole-genome scale and in a quantitative context. In C. ...

High-throughput Purification of Affinity-tagged Recombinant Proteins

X-ray crystallography is the method of choice for obtaining a detailed view of the structure of proteins. Such studies need to be complemented by further biochemical analyses to obtain detailed insights into structure/function relationships. Advances in oligonucleotide- and gene synthesis technology make large-scale mutagenesis strategies increasingly feasible, including the substitution of target residues by all 19 other amino acids. Gain- or loss-of-function phenotypes then allow systematic conclusions to be drawn, such as the contribution of particular residues to catalytic activity, protein stability and/or protein-protein interaction specificity.
In order to attribute the different phenotypes to the nature of the mutation - rather than to fluctuating experimental conditions - it is vital to purify and analyse the proteins in a controlled and reproducible manner. High-throughput strategies and the automation of manual protocols on robotic liquid-handling platforms have created opportunities to perform such complex molecular biological procedures with little human intervention and minimal error rates1-5.
Here, we present a general method for the purification of His-tagged recombinant proteins in a high-throughput manner. In a recent study, we applied this method to a detailed structure-function investigation of TFIIB, a component of the basal transcription machinery. TFIIB is indispensable for promoter-directed transcription in vitro and is essential for the recruitment of RNA polymerase into a preinitiation complex6-8. TFIIB contains a flexible linker domain that penetrates the active site cleft of RNA polymerase9-11. This linker domain confers two biochemically quantifiable activities on TFIIB, namely (i) the stimulation of the catalytic activity during the 'abortive' stage of transcript initiation, and (ii) an additional contribution to the specific recruitment of RNA polymerase into the preinitiation complex4,5,12 . We exploited the high-throughput purification method to generate single, double and triple substitution and deletions mutations within the TFIIB linker and to subsequently analyse them in functional assays for their stimulation effect on the catalytic activity of RNA polymerase4. Altogether, we generated, purified and analysed 381 mutants - a task which would have been time-consuming and laborious to perform manually. We produced and assayed the proteins in multiplicates which allowed us to appreciate any experimental variations and gave us a clear idea of the reproducibility of our results.
This method serves as a generic protocol for the purification of His-tagged proteins and has been successfully used to purify other recombinant proteins. It is currently optimised for the purification of 24 proteins but can be adapted to purify up to 96 proteins.

We describe a method for the affinity-tagged purification of recombinant proteins using liquid-handling robotics. This method is generally applicable to the small-scale purification of soluble His-tagged proteins in a high-throughput format.

Affinity etiketli Rekombinant Proteinler Yüksek throughput Arıtma

X-ray crystallography is the method of choice for obtaining a detailed view of the structure of proteins. Such studies need to be complemented by further ...

High-throughput Screening for Small-molecule Modulators of Inward Rectifier Potassium Channels

Specific members of the inward rectifier potassium (Kir) channel family are postulated drug targets for a variety of disorders, including hypertension, atrial fibrillation, and pain1,2. For the most part, however, progress toward understanding their therapeutic potential or even basic physiological functions has been slowed by the lack of good pharmacological tools. Indeed, the molecular pharmacology of the inward rectifier family has lagged far behind that of the S4 superfamily of voltage-gated potassium (Kv) channels, for which a number of nanomolar-affinity and highly selective peptide toxin modulators have been discovered3. The bee venom toxin tertiapin and its derivatives are potent inhibitors of Kir1.1 and Kir3 channels4,5, but peptides are of limited use therapeutically as well as experimentally due to their antigenic properties and poor bioavailability, metabolic stability and tissue penetrance. The development of potent and selective small-molecule probes with improved pharmacological properties will be a key to fully understanding the physiology and therapeutic potential of Kir channels.
The Molecular Libraries Probes Production Center Network (MLPCN) supported by the National Institutes of Health (NIH) Common Fund has created opportunities for academic scientists to initiate probe discovery campaigns for molecular targets and signaling pathways in need of better pharmacology6. The MLPCN provides researchers access to industry-scale screening centers and medicinal chemistry and informatics support to develop small-molecule probes to elucidate the function of genes and gene networks. The critical step in gaining entry to the MLPCN is the development of a robust target- or pathway-specific assay that is amenable for high-throughput screening (HTS).
Here, we describe how to develop a fluorescence-based thallium (Tl+) flux assay of Kir channel function for high-throughput compound screening7,8,9,10.The assay is based on the permeability of the K+ channel pore to the K+ congener Tl+. A commercially available fluorescent Tl+ reporter dye is used to detect transmembrane flux of Tl+ through the pore. There are at least three commercially available dyes that are suitable for Tl+ flux assays: BTC, FluoZin-2, and FluxOR7,8. This protocol describes assay development using FluoZin-2. Although originally developed and marketed as a zinc indicator, FluoZin-2 exhibits a robust and dose-dependent increase in fluorescence emission upon Tl+ binding. We began working with FluoZin-2 before FluxOR was available7,8 and have continued to do so9,10. However, the steps in assay development are essentially identical for all three dyes, and users should determine which dye is most appropriate for their specific needs. We also discuss the assay's performance benchmarks that must be reached to be considered for entry to the MLPCN. Since Tl+ readily permeates most K+ channels, the assay should be adaptable to most K+ channel targets.

Methods for developing and validating a quantitative fluorescence assay for measuring the activity of inward rectifier potassium (Kir) channels for high-throughput compound screening is presented.

İçte düzeltici Potasyum Kanallarının Küçük moleküllü Modülatörler için Yüksek throughput Eleme

Specific members of the inward rectifier potassium (Kir) channel family are postulated drug targets for a variety of disorders, including hypertension, ...

C. elegans Chemotaxis Assay

Many organisms use chemotaxis to seek out food sources, avoid noxious substances, and find mates. Caenorhabditis elegans has impressive chemotaxis behavior.
 The premise behind testing the response of the worms to an odorant is to place them in an area and observe the movement evoked in response to an odorant. Even with the many available assays, optimizing worm starting location relative to both the control and test areas, while minimizing the interaction of worms with each other, while maintaining a significant sample size remains a work in progress 1-10. The method described here aims to address these issues by modifying the assay developed by Bargmann et al.1. A Petri dish is divided into four quadrants, two opposite quadrants marked "Test" and two are designated "Control". Anesthetic is placed in all test and control sites. The worms are placed in the center of the plate with a circle marked around the origin to ensure that non-motile worms will be ignored. Utilizing a four-quadrant system rather than one 2 or two 1 eliminates bias in the movement of the worms, as they are equidistant from test and control samples, regardless of which side of the origin they began. This circumvents the problem of worms being forced to travel through a cluster of other worms to respond to an odorant, which can delay worms or force them to take a more circuitous route, yielding an incorrect interpretation of their intended path. This method also shows practical advantages by having a larger sample size and allowing the researcher to run the assay unattended and score the worms once the allotted time has expired.

C. elegans Chemotaxis Assay

A method of quantitatively evaluating the chemotactic response of Caenorhabditis elegans is described. A chemotactic index (CI) was employed as a way to precisely evaluate the response of worms to certain targets, and serve as a platform of comparison between strains and compounds of interest.

C. elegans Kemotaksis Deneyi

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

Biochemical and High Throughput Microscopic Assessment of Fat Mass in Caenorhabditis Elegans

The nematode C. elegans has emerged as an important model for the study of conserved genetic pathways regulating fat metabolism as it relates to human obesity and its associated pathologies. Several previous methodologies developed for the visualization of C. elegans triglyceride-rich fat stores have proven to be erroneous, highlighting cellular compartments other than lipid droplets. Other methods require specialized equipment, are time-consuming, or yield inconsistent results. We introduce a rapid, reproducible, fixative-based Nile red staining method for the accurate and rapid detection of neutral lipid droplets in C. elegans. A short fixation step in 40% isopropanol makes animals completely permeable to Nile red, which is then used to stain animals. Spectral properties of this lipophilic dye allow it to strongly and selectively fluoresce in the yellow-green spectrum only when in a lipid-rich environment, but not in more polar environments. Thus, lipid droplets can be visualized on a fluorescent microscope equipped with simple GFP imaging capability after only a brief Nile red staining step in isopropanol. The speed, affordability, and reproducibility of this protocol make it ideally suited for high throughput screens. We also demonstrate a paired method for the biochemical determination of triglycerides and phospholipids using gas chromatography mass-spectrometry. This more rigorous protocol should be used as confirmation of results obtained from the Nile red microscopic lipid determination. We anticipate that these techniques will become new standards in the field of C. elegans metabolic research.

Biochemical and High Throughput Microscopic Assessment of Fat Mass in Caenorhabditis Elegans

We present robust biochemical and microscopic methods for studying Caenorhabditis elegans lipid stores. A rapid, simple, fixing-staining procedure for fluorescent lipid droplet imaging leverages the spectral properties of the lipophilic dye Nile red. We then present biochemical measurement of triglycerides and phospholipids using solid phase extraction and gas chromatography-mass spectrometry.

Fat Kütle Biyokimyasal ve Yüksek Verimli Mikroskobik Değerlendirme<em&gt; Caenorhabditis Elegans</em

The nematode C. elegans has emerged as an important  model for the study of conserved genetic pathways regulating fat metabolism as  it relates to human ...

High-throughput Functional Screening using a Homemade Dual-glow Luciferase Assay

We present a rapid and inexpensive high-throughput screening protocol to identify transcriptional regulators of alpha-synuclein, a gene associated with Parkinson&#39;s disease. 293T cells are transiently transfected with plasmids from an arrayed ORF expression library, together with luciferase reporter plasmids, in a one-gene-per-well microplate format. Firefly luciferase activity is assayed after 48 hr to determine the effects of each library gene upon alpha-synuclein transcription, normalized to expression from an internal control construct (a hCMV promoter directing Renilla luciferase). This protocol is facilitated by a bench-top robot enclosed in a biosafety cabinet, which performs aseptic liquid handling in 96-well format. Our automated transfection protocol is readily adaptable to high-throughput lentiviral library production or other functional screening protocols requiring triple-transfections of large numbers of unique library plasmids in conjunction with a common set of helper plasmids. We also present an inexpensive and validated alternative to commercially-available, dual luciferase reagents which employs PTC124, EDTA, and pyrophosphate to suppress firefly luciferase activity prior to measurement of Renilla luciferase. Using these methods, we screened 7,670 human genes and identified 68 regulators of alpha-synuclein. This protocol is easily modifiable to target other genes of interest.

We present a rapid and inexpensive screening method for identifying transcriptional regulators using high-throughput robotic transfections and a homemade dual-glow luciferase assay. This protocol rapidly generates direct side-by-side functional data for thousands of genes and is easily modifiable to target any gene of interest.

Bir ev yapımı Çift kızdırma Lusıferaz Testi kullanarak yüksek verim Fonksiyonel Taraması

We present a rapid and inexpensive high-throughput screening protocol to identify transcriptional regulators of alpha-synuclein, a gene associated with ...

High-throughput Screening for Chemical Modulators of Post-transcriptionally Regulated Genes

Both transcriptional and post-transcriptional regulation have a profound impact on genes expression. However, commonly adopted cell-based screening assays focus on transcriptional regulation, being essentially aimed at the identification of promoter-targeting molecules. As a result, post-transcriptional mechanisms are largely uncovered by gene expression targeted drug development. Here we describe a cell-based assay aimed at investigating the role of the 3&#39; untranslated region (3&#8217; UTR) in the modulation of the fate of its mRNA, and at identifying compounds able to modify it. The assay is based on the use of a luciferase reporter construct containing the 3&#8217; UTR of a gene of interest stably integrated into a disease-relevant cell line. The protocol is divided into two parts, with the initial focus on the primary screening aimed at the identification of molecules affecting luciferase activity after 24 hr of treatment. The second part of the protocol describes the counter-screening necessary to discriminate compounds modulating luciferase activity specifically through the 3&#8217; UTR. In addition to the detailed protocol and representative results, we provide important considerations about the assay development and the validation of the hit(s) on the endogenous target. The described cell-based reporter gene assay will allow scientists to identify molecules modulating protein levels via post-transcriptional mechanisms dependent on a 3&#8217; UTR.

Here we describe a cell-based reporter gene assay as a valuable tool to screen chemical libraries for compounds modulating post-transcriptional control mechanisms exerted through 3&#8217; UTR.

Post-transkripsiyonel ayarlı genlerin kimyasal Modülatörler için yüksek verimli tarama

Both transcriptional and post-transcriptional regulation have a profound impact on genes expression. However, commonly adopted cell-based screening assays ...

Identification of Kinase-substrate Pairs Using High Throughput Screening

We have developed a screening platform to identify dedicated human protein kinases for phosphorylated substrates which can be used to elucidate novel signal transduction pathways. Our approach features the use of a library of purified GST-tagged human protein kinases and a recombinant protein substrate of interest. We have used this technology to identify MAP/microtubule affinity-regulating kinase 2 (MARK2) as the kinase for a glucose-regulated site on CREB-Regulated Transcriptional Coactivator 2 (CRTC2), a protein required for beta cell proliferation, as well as the Axl family of tyrosine kinases as regulators of cell metastasis by phosphorylation of the adaptor protein ELMO. We describe this technology and discuss how it can help to establish a comprehensive map of how cells respond to environmental stimuli.

Protein phosphorylation is a central feature of how cells interpret and respond to information in their extracellular milieu. Here, we present a high throughput screening protocol using kinases purified from mammalian cells to rapidly identify kinases that phosphorylate a substrate(s) of interest.

Yüksek Verimli Tarama kullanma Kinaz-substrat Çiftlerinin Kimlik


We have developed a screening platform to identify dedicated human protein kinases for phosphorylated substrates which can be used to elucidate novel ...