Bu çalışma Sağlık MH097163 National Institutes fonları tarafından desteklenmiştir. Bazı suşlar Araştırma Altyapı Programları NIH Ofisi (P40-OD010440) tarafından finanse CGC tarafından sağlanmıştır.

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Yazarlar, hiçbir rakip mali çıkarlarını olmadığını beyan ederim.

Biz C. büyük ölçekli ekranlar gerçekleştirmek için piyasada mevcut DsRNA besleme kitaplığı kullanan bir protokol tanımlanmıştır elegans. Biz kütüphane çoğaltmak ve etkili bir şekilde kullanmak için tüm talimatları sağlar. Bu yaklaşım, hayvanın farklı dokularda farklı biyolojik proseslerde yer alan genleri tanımlamak mümkün olduğunu göstermektedir. Burada tarif fenotipleri (UNC, Egl ve dpy) kolayca gözlemlenebilir olsa da, bu protokol, hemen hemen herhangi bir biyolojik...

C. Tarihsel olarak, genetik ekranlar elegans DNA içinde rasgele mutasyona oluşturmak için, örneğin etil metansülfonat gibi bir kimyasal mutajen ile hayvanların tedavi edilmesi ile gerçekleştirilmiştir. Mutagenize edilmiş hayvanların soyu veya grandprogeny sonra sergilediğini arzu edilen bir anormal bir fenotip izole edilir. Fenotipe neden sorumlu mutasyon daha sonra bir emek-yoğun bir eşleme prosedürü ile ya da mutant solucan tüm genom sekanslama ile tanımlanır. Orada, kazanım fonksiyon-of ve zarar fonksiyon-fenotipler neden olabilir solucan genomu içinde kalıcı lezyonlar oluşturmak gibi kimyasal mutagenez ekranlar performans için pek çok avantajı vardır, ancak eşleme prosedürü yavaş ve pahalıdır. Çift kollu bir RNA girişimi (dsRNAi) önemli biyolojik proseslerde yer alan genleri tanımlamak için alternatif bir yol sağlar. Bu yöntemde, bir endojen genin kodlama dizisi eşleşen dsRNA solucan sokulur.DsRNA kılavuzları yok 1 için onları hedef, uygun endojen mRNA bulmak ve bağlamak üzere görev küçük (21-22 nükleotid) RNA&#39;ları oluşturmak için in vivo olarak işlenir. Bu prosedür nispeten hızlı olduğunu, ancak dsRNA&#39;nın yerine DNA&#39;dan daha mRNA hedefleri nedeniyle, sadece azaltma fonksiyon-fenotipleri bu yaklaşımı kullanılarak gözlenmiştir. Bir hayvana dsRNA&#39;lar sokulması dsRNA 3 hayvanları ıslatarak ya da 4 dsRNA eksprese hayvan bakteri beslenmesiyle, dsRNA 2 direkt enjeksiyonu ile elde edilebilir. Hayvanın en hücreleri SID-1, 5 dsRNA&#39;yı ithal edebilen bir transmembran kanalı ifade olarak bu teslim yöntemlerin her sistemik devirme etkilere neden olabilir. Başlangıçta her seferinde tek tek genlerin ekspresyonunu bir demonte bir karşıt genetik yaklaşım olarak, iki besleme kütüphanelerinin oluşturulmasına şimdi büyük ölçekli genetik ekranlar izin verir. Piyasada bulunan DsRNA kütüphaneleri ba içerirlerTüm C.% 55 veya% 87 ya da temsil cterial klonlar elegans genleri 6, 7. Ne yazık ki, büyük ölçekli dsRNAi besleme ekranlar genellikle değişken devirme verimliliği 8-10 sonuçlanabilir. RNAi Nakavt mutlak seviyesi kolaylıkla ölçülebilir olmasa da, büyük ölçekli ekranlar gözlenen etkinlik yok etme, indirgenmiş RNAi tarafından bozulmasını kaçış kalıntı gene özgü mRNA neden olmalıdır. Bu da, bu ekranlarında hayvanlara yem bakterilerden dsRNA plasmid kaybı doğrudan bir sonucu olabilir. Demonte etkileri beslenen hayvanlara 11 kontrol ile karşılaştırıldığında (eğer varsa) bu fikri desteklemek, hayvanlar bakterinin sadece% 50 bir hedef gene karşı dsRNA&#39;lar ifade ettiği, bakteri karışımları beslenen, önemli ölçüde azalır göstermektedir. C. çoğu genler olarak dsRNAi ekranlarında yaparken gen Nakavt ölçüde kritik bir endişe olur elegans haplosufficient ve böylece gen tanımı, en az% 50 t azaltılmalıdıro kayıp fonksiyon-12 fenotip neden olur. Biz büyük ölçekli ekranlar gerçekleştirmek için daha önceden yayınlanmış besleme protokolleri ve diğerleri 8-10 tarafından bildirilen aynı değişken devirme etkileri bulduk. Olası nedenler için bir arama, biz ekranda hayvanlara beslenen bakterilerin yaklaşık% 60 dsRNA&#39;nın plazmid kaybetmişti bulundu. Biz dramatik azaltır veya ortadan kaldırır plazmid kaybı ve büyük ölçüde devirme etkinliğini arttırır kütüphane çoğaltma ve tarama protokolü geliştirdik. Bu protokol C de büyük ölçekli ekranlar yapmak için uygundur elegans ve ticari olarak temin edilebilen dsRNA kitaplıkları birini ya da taranması için kullanılabilir. Bu protokolü kullanarak, ekrana sırasında hayvanların beslenen bakterilerin esasen% 100 dsRNA kodlayan plazmid korumak ve, incelenen tüm dokularda işlev fenotipleri sağlam ve yüksek nüfuz kaybına yol bulmak.

<table border="1">
 <tbody>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>Reagent/Material</td>
 </tr>
 <tr>
 <td>96-well Falcon flat bottom plate </td>
 <td>Fisher Scientific</td>
 <td>353072</td>
 <td>sterile</td>
 </tr>
 <tr>
 <td>adhesive foil</td>
 <td>USA Scientific</td>
 <td>2923-0100</td>
 <td></td>
 </tr>
 <tr>
 <td>Nunc omniplate</td>
 <td>Fisher Scientific</td>
 <td>267060</td>
 <td></td>
 </tr>
 <tr>
 <td>2.0 ml deep 96-well polypropylene Masterblock</td>
 <td>USA Scientific</td>
 <td>5678-0285</td>
 <td>autoclavable</td>
 </tr>
 <tr>
 <td>Lids for deep well plates</td>
 <td>USA Scientific</td>
 <td>5665-6101</td>
 <td></td>
 </tr>
 <tr>
 <td>50 ml combitips</td>
 <td>USA Scientific</td>
 <td>4796-5000</td>
 <td>individually wrapped</td>
 </tr>
 <tr>
 <td>Reservoir</td>
 <td>USA Scientific</td>
 <td>2977-8500</td>
 <td>autoclavable</td>
 </tr>
 <tr>
 <td>12-well plates</td>
 <td>USA Scientific</td>
 <td>CC7682-7512</td>
 <td>individually wrapped</td>
 </tr>
 <tr>
 <td>dsRNA feeding library</td>
 <td>OpenBiosystems</td>
 <td>RCE1181</td>
 <td>store -80 &deg;C</td>
 </tr>
 <tr>
 <td>Ampicillin</td>
 <td>Fisher Scientific</td>
 <td>BP1760-5</td>
 <td></td>
 </tr>
 <tr>
 <td>Tetracycline</td>
 <td>Fisher Scientific</td>
 <td>BP912-100</td>
 <td></td>
 </tr>
 <tr>
 <td>Carbenicillin</td>
 <td></td>
 <td></td>
 <td>allow 2-4 weeks for delivery <a href="http://www.biochemicaldirect.com" target="_blank">www.biochemicaldirect.com</a></td>
 </tr>
 <tr>
 <td>Bacteriolgical Agar Ultrapure grade A </td>
 <td>Affymetrix</td>
 <td>10906</td>
 <td>for worm plates</td>
 </tr>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>Equipment</td>
 </tr>
 <tr>
 <td>Brayer roller</td>
 <td>USA Scientific</td>
 <td>9127-2940</td>
 <td></td>
 </tr>
 <tr>
 <td>Boekel 96-pin replicator</td>
 <td>Fisher Scientific</td>
 <td>05-450-9</td>
 <td>autoclavable</td>
 </tr>
 <tr>
 <td>microshaker</td>
 <td>Thomas Scientific</td>
 <td>1231A93</td>
 <td>3 mm orbit</td>
 </tr>
 <tr>
 <td>12 channel multichannel pipet (0.5-10 &mu;l)</td>
 <td>USA Scientific</td>
 <td>7112-0510</td>
 <td></td>
 </tr>
 <tr>
 <td>12 channel multichannel pipet (30-300 &mu;l)</td>
 <td>USA Scientific</td>
 <td>7112-3300</td>
 <td></td>
 </tr>
 <tr>
 <td>Repeater Plus manual pipettor</td>
 <td>USA Scientific</td>
 <td>4026-0201</td>
 <td></td>
 </tr>
 </tbody>
 </table>

large-scale gene knockdown in c. elegans using dsrna feeding libraries to generate robust loss-of-function phenotypes

DsRNAlar ifade solucanlar bakterileri besleyerek RNA girişim C gen fonksiyonunu değerlendirmek için yararlı bir araç olmuştur elegans. Genlerin küçük bir sayıda demonte için hedeflenen bu stratejisi iyi çalışıyor olsa da, büyük ölçekli besleme ekranları kendi programını sınırlar, değişken knockdown verimliliğini gösterir. Daha önce yayınlanan RNAi yok etme protokolleri deconstructed ve indirgenmiş Nakavt birincil kaynağı hayvanlara beslenen bakterilerden dsRNA kodlayan plazmidlerin kaybına bağlı olabilir bulduk. Bu gözlemlere dayanarak, biz büyük ölçüde azaltır veya verimli, yüksek verim elde etmek için demonte plazmid kaybı ortadan kaldıran bir DsRNA besleme protokol geliştirdik. Biz bu protokol C. aşağı sağlam, tekrarlanabilir darbe üretecek olduğunu göstermek nöronlar elegans da dahil olmak üzere çok sayıda doku türleri içinde, genler, ve büyük ölçekli ekranlarında etkili demonte izin verecektir. Bu protokol, bir ticari olarak temin edilebilir besleme dsRNA kullanankütüphane ve kütüphane çoğaltmak ve DsRNA ekranları gerçekleştirmek için gereken tüm adımları açıklar. Protokol, bir karmaşık ekipman kullanımını gerektirmez ve bu nedenle herhangi bir C. tarafından yapılabilir laboratuvar elegans.

P0 knockdown fenotipleri DsRNA bakterileri ifade hayvanların beslenmesi 2-3 gün sonra görünmeye başlayacaktır. Bazı erken fenotipleri larva tutuklama ve öldürücülüğü içerir ve tüm beslenme kuyuların% 2-3 dikkat edilmelidir. Bu aynı fenotipleri de F1 üretimi hayvanlarda gözlenecektir. Yumurtadan başarısız F1 yumurta varlığı başka ortak F1 fenotip ve birlikte, bu üç fenotipleri F1 ekranlarında tüm kuyuların yaklaşık 10% olarak görülecektir. Biz P0 ve F1 feno...

Watch this Scientific Journal Video about Large-scale Gene Knockdown in C. elegans Using dsRNA Feeding Libraries to Generate Robust Loss-of-function Phenotypes at JoVE.com

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

DsRNA C. beslerken elegans büyük ölçekli besleme ekranlar için geçerli protokoller değişken devirme verimleri elde gen fonksiyonunu değerlendirmek için güçlü bir araçtır. Bu gen ifadesinin oldukça etkili ve yeniden üretilebilir bir devirme ile sonuçlanan büyük ölçekli RNAi besleme ekranları yerine getirmek için geliştirilmiş bir protokol açıklar.

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 ...

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Biology

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

11.9K Görüntüleme.  University of Massachusetts, Amherst.  DsRNA C. beslerken elegans büyük ölçekli besleme ekranlar için geçerli protokoller değişken devirme verimleri elde gen fonksiyonunu değerlendirmek için güçlü bir araçtır. Bu gen ifadesinin oldukça etkili ve yeniden üretilebilir bir devirme ile sonuçlanan büyük ölçekli RNAi besleme ekranları yerine getirmek için geliştirilmiş bir protokol açıklar. 

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

Large-Scale Screens of Metagenomic Libraries

Metagenomic libraries archive large fragments of contiguous genomic sequences from microorganisms without requiring prior cultivation. Generating a streamlined procedure for creating and screening metagenomic libraries is therefore useful for efficient high-throughput investigations into the genetic and metabolic properties of uncultured microbial assemblages. Here, key protocols are presented on video, which we propose is the most useful format for accurately describing a long process that alternately depends on robotic instrumentation and (human) manual interventions. First, we employed robotics to spot library clones onto high-density macroarray membranes, each of which can contain duplicate colonies from twenty-four 384-well library plates. Automation is essential for this procedure not only for accuracy and speed, but also due to the miniaturization of scale required to fit the large number of library clones into highly dense spatial arrangements. Once generated, we next demonstrated how the macroarray membranes can be screened for genes of interest using modified versions of standard protocols for probe labeling, membrane hybridization, and signal detection. We complemented the visual demonstration of these procedures with detailed written descriptions of the steps involved and the materials required, all of which are available online alongside the video.

Metagenomic Kütüphaneler Büyük Ölçekli Ekranlar

Metagenomic libraries archive large fragments of contiguous genomic sequences from microorganisms without requiring prior cultivation.  Generating a ...

Biyoloji

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 ...

Using an Automated Cell Counter to Simplify Gene Expression Studies: siRNA Knockdown of IL-4 Dependent Gene Expression in Namalwa Cells

The use of siRNA mediated gene knockdown is continuing to be an important tool in studies of gene expression.  siRNA studies are being conducted not only to study the effects of downregulating single genes, but also to interrogate signaling pathways and other complex interaction networks.  These pathway analyses require both the use of relevant cellular models and methods that cause less perturbation to the cellular physiology.  Electroporation is increasingly being used as an effective way to introduce siRNA and other nucleic acids into difficult to transfect cell lines and primary cells without altering the signaling pathway under investigation. There are multiple critical steps to a successful siRNA experiment, and there are ways to simplify the work while improving the data quality at several experimental stages.  To help you get started with your siRNA mediated gene knockdown project, we will demonstrate how to perform a pathway study complete from collecting and counting the cells prior to electroporation through post transfection real-time PCR gene expression analysis.  The following study investigates the role of the transcriptional activator STAT6 in IL-4 dependent gene expression of CCL17 in a Burkitt lymphoma cell line (Namalwa).  The techniques demonstrated are useful for a wide range of siRNA-based experiments on both adherent and suspension cells.  We will also show how to streamline cell counting with the TC10 automated cell counter, how to electroporate multiple samples simultaneously using the MXcell electroporation system, and how to simultaneously assess RNA quality and quantity with the Experion automated electrophoresis system.

This procedure describes a quick and easy workflow to introduce siRNA into difficult to transfect cell lines and follow gene expression by real-time PCR. Use of an automated cell counter, multi-well electroporation plate, and automated electrophoresis station provide quick and reliable results without the need for expensive robotic handling.

Gen İfadesi Çalışmaları basitleştirin Otomatik Hücre Sayıcı kullanma: Namalwa Hücreler IL-4 Bağımlı Gen İfadesi siRNA demonte

The use of siRNA mediated gene knockdown is continuing to be an important tool in studies of gene expression.  siRNA studies are being conducted not only ...

Large Scale Zebrafish-Based In vivo Small Molecule Screen

Given their small embryo size, rapid development, transparency, fecundity, and numerous molecular, morphological and physiological similarities to mammals, zebrafish has emerged as a powerful in vivo platform for phenotype-based drug screens and chemical genetic analysis. Here, we demonstrate a simple, practical method for large-scale screening of small molecules using zebrafish embryos.

Large Scale Zebrafish-Based In vivo Small Molecule Screen

Zebrafish has emerged as a powerful in vivo platform for phenotype-based drug screens and chemical genetic analysis. Here, we demonstrate a simple, practical method for large-scale screening of small molecules using zebrafish embryos.

Büyük Ölçekli Zebra balığı Tabanlı In vivo Küçük Molekül Ekran

Given their small embryo size, rapid development, transparency, fecundity, and numerous molecular, morphological and physiological similarities to ...

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 ...

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 ...

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

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 including plants, invertebrates and vertebrates. There are various methods to achieve RNAi in vivo4,5. For example, the target gene may be transformed into an RNAi vector, and then either permanently or transiently transformed into cell lines or primary cells to achieve gene knockdown effects; alternatively synthesized double-strand oligonucleotides from specific target genes (RNAi oligos) may be transiently transformed into cell lines or primary cells to silence target genes; or synthesized double-strand RNA molecules may be microinjected into an organism. Since the nematode C. elegans uses bacteria as a food source, feeding the animals with bacteria expressing double-strand RNA against target genes provides a viable strategy6. Here we present an RNAi feeding method to score body size phenotype. Body size in C. elegans is regulated primarily by the TGF- &beta; - like ligand DBL-1, so this assay is appropriate for identification of TGF-&beta; signaling components7. We used different strains including two RNAi hypersensitive strains to repeat the RNAi feeding experiments. Our results showed that rrf-3 strain gave us the best expected RNAi phenotype. The method is easy to perform, reproducible, and easily quantified. Furthermore, our protocol minimizes the use of specialized equipment, so it is suitable for smaller laboratories or those at predominantly undergraduate institutions.

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

We demonstrate how to use the RNAi feeding technique to knock down target genes and score body size phenotype in C. elegans. This method could be used for a large scale screen to identify potential genetic components of interest, such as those involved in body size regulation by DBL-1/TGF-&beta; signaling.

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 ...

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 ...

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 ...

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 ...