Nous sommes particulièrement reconnaissants à Seth Darst pour fournir des protéines ARNP et MFD et vecteurs d&#39;expression. Nous sommes également à remercier Sergueï Borukhov pour fournir Greb et Dan Zhang pour le soutien technique. Ce travail a été soutenu par une subvention des National Institutes of Health (MOD) et par une Marie-Josée et Henry Kravis Fellowship à l&#39;Université Rockefeller (RTP).

Cette méthode a été utilisée dans la recherche présentée dans <a href="http://www.sciencemag.org/cgi/content-nw/full/327/5965/590/F3">Pomerantz et O&#39;Donnell, Science 327, 590-592 (2010)</a> . 1. Assemblée d&#39;un complexe d&#39;élongation a été stoppé ARNP effectuée comme suit: 500 nM de concentration finale de E. ARNP coli σ 70 ans, il a été mélangé avec une concentration de 5 nM final d&#39;une matrice d&#39;ADN biotinylé 3,6 kb contenant le promoteur T7A1 dans 100 ul de tampon A (20 mM Tris-Cl (pH 7,5), 8 mM de MgCl2, 0,5 mM EDTA, 5 mM DTT, 10% glycérol) pendant 10 min à 37 ° C. 100 uM de l&#39;APU et 40 uM de chacun de GTP, ATP, et CTP ont été ajoutés pour encore 10 min à 37 ° C. 2. Immobilisation du complexe d&#39;élongation ARNP stoppé sur des billes de streptavidine a été effectuée comme suit: 200 ul de streptavidine billes magnétiques revêtues (Invitrogen) ont été ajoutés à la réaction ci-dessus pendant 10 min à température ambiante. 3. Purification du complexe immobilisé ARNP stoppé l&#39;élongation a été effectuée comme suit: Les perles (en haut) ont été lavées 5 fois avec 0,9 ml de tampon A contenant 0,75 M de NaCl, 200 ug / ml d&#39;héparine, et 20 pg / ml ADNss (Le surnageant a été éliminé après chaque étape de lavage par séparation magnétique.) Ensuite, le billes ont été lavées deux fois avec 0,9 ml de tampon A. 4. Formation d&#39;une fourche de réplication en aval par rapport à la tête sur ARNP a été effectuée comme suit: Les perles (en haut) ont été remises en suspension dans 100 ul de tampon de la Nouvelle Angleterre Biolabs 4 et 10 unités de Sap I (New England Biolabs) a été ajouté pendant 10 min à 37 ° C. Les billes ont été lavées trois fois avec 0,9 ml de tampon A puis remis en suspension dans 50 pl de tampon de ligation réaction rapide T4 (New England Biolabs). 2 pi de ligase T4 rapide (New England Biolabs) a été ajouté avec 6 concentrations nM finale de pré-recuit ADN fourchue (RP10, RP22, RP25) pendant 10 min à température ambiante. Les billes ont été lavées trois fois avec 0,9 ml de tampon A. 5. Assemblée de l&#39;réplisome et l&#39;initiation de la synthèse des brins de premier plan à la fourche de réplication a été effectuée comme suit: 448 pmol de DnaB (comme hexamère) a été incubée avec les 150 perles ul de tampon A (20 mM de glycérol Tris-Cl (pH 7,5), 8 mM de MgCl2, 0,5 mM EDTA, 5 mM DTT, 10%) pendant 30 s à 23 ° C. 4.9 pmol de Pol III * (Pol III, il minus β), 15 pmol de β, ATP 2 mM, et 60 uM chacun de dGTP et dATP ont été ajoutés à un volume de 200 ul et incubée encore 5 min à 37 ° C. La réplication a été lancé lors de l&#39;ajout de 10 mg et SSB 24 uM de chacun de dATP et dTTP avec [α-32 P] dTTP et [α-32 P] dCTP (activité spécifique, 3000-5000 cpm / pmol) pour un volume final de 250 ul. Réactions ont été interrompues au bout de 10 min après addition de 12 ul d&#39;EDTA 500 mM. 6. Purification et la concentration de la radio-labellisés ADN a été effectuée comme suit: Les perles (en haut) ont été cuits après la réaction a été arrêtée afin d&#39;éliminer l&#39;ADN à partir des perles. Toute l&#39;ADN résiduel restant attaché à billes a été éliminé en traitant les perles avec la protéinase K dans un volume de 10 ul pendant 30 min à 50 ° C. Les billes sont ensuite bouillies et le surnageant a été enlevé par séparation magnétique. Le surnageant contenant l&#39;ADN combinée a été purifié en utilisant le kit Qiagen PCR Cleanup. Purifié radiomarqué produits d&#39;ADN ont ensuite été analysés dans un gel d&#39;agarose alcalines. Réplication * en l&#39;absence d&#39;une tête sur ARNP stoppé complexe d&#39;élongation a été effectuée comme ci-dessus, cependant, σ 70 a été omise. Les résultats représentatifs: Collision de l&#39;réplisome avec une tête sur ARNP se traduit généralement par deux produits de 2,5 kb et 3,6 kb de longueur. Le produit de 2,5 kb représente la longueur de l&#39;ADN de la fourche à la ARNP stoppé et les résultats de réplisome décrochage lors d&#39;une collision avec le ARNP. Le produit 3,6 kb représentent pleine longueur de l&#39;ADN et les résultats de l&#39;occupation soit incomplète du promoteur par ARNP ou partielle réplisome lecture par le biais de l&#39;ARNP front. Un bon résultat ou précis démontre qu&#39;environ 50% de la pleine-longueur de l&#39;ADN est produit. Toutefois, dans certains cas, moins d&#39;occupation du promoteur par ARNP se produit et un pourcentage plus élevé de toute la longueur ADN est observée depuis une plus grande population de replisomes ne rencontrent pas une ARNP front. Réplication en l&#39;absence d&#39;un résultat ARNP stoppé en seulement plein longueur de l&#39;ADN (3,6 kb). <img src="/files/ftp_upload/1919/1919fig1.jpg" alt="Figure 1" /> Figure. 1 Le réplisome est entravée par un ARNP front. (A) Montage expérimental. Un E. Coli ARNP complexe d&#39;élongation arrêté a été assemblé sur un ADN biotinylé contenant le promoteur T7A1 comme décrit précédemment (9). Brièvement, un complexe d&#39;élongation arrêté a été formé 20 bps aval du promoteur en incubant holoenzyme ARNP avec l&#39;ADN biotinylé pendant 10 min suivi par l&#39;ajout d&#39;Apu, le GTP, ATP, CTP et pendant encore 10 min. Billes de streptavidine ont été ajoutés pour encore 10 min puis les billes ont été lavées cinq fois chacune avec 0,9 ml de tampon contenant 0,75 M de NaCl, 200 mg / ml d&#39;héparine, et 100 nM seul brin d&#39;ADN pour retirer instables ARNP complexes ADN-et PNT excès . L&#39;ADN a ensuite été digéré avec Sapi, lavées, et ligué à un ADN pré-recuit fourchue. Les billes ont été lavées à nouveau avant l&#39;assemblage du réplisome. Le promoteur est situé à 2,5 kb de la fourche et est orientée à la transcription directe vers la fourche (B). DnaB a été ajouté puis le réplisome a été assemblé et la réplication a été initié, soit en présence (piste 2) ou l&#39;absence (piste 1) d&#39;un front ARNP.

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W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Twin+DNA+pumps+of+a+hexameric+helicase+provide+power+to+simultaneously+melt+two+duplexes.">Twin DNA pumps of a hexameric helicase provide power to simultaneously melt two duplexes.</a> Cell. 109, 757-757 (2002).</li><li>Hanawalt, P. C., Spivak, G. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=coli+Transcription+repair+coupling+factor+(Mfd+protein)+rescues+arrested+complexes+by+promoting+forward+translocation.">coli Transcription repair coupling factor (Mfd protein) rescues arrested complexes by promoting forward translocation.</a> Nat Rev Mol Cell Biol. 9, 958-958 (2008).</li><li>Hanawalt, P. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transcription-coupled+DNA+repair:+two+decades+of+progress+and+surprises.">Transcription-coupled DNA repair: two decades of progress and surprises.</a> Science. 266, 1957-1957 (1994).</li><li>Selby, C. P., Sancar, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transcription-coupled+repair+and+human+disease.">Transcription-coupled repair and human disease.</a> J Biol Chem. 270, 4882-4882 (1995).</li><li>Liu, B., Alberts, B. 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Le lavage du complexe d&#39;élongation stoppé lié aux billes avec du sel élevée est essentielle afin de supprimer tout instable ou non spécifiquement molécules ARNP lié. ARNP a une affinité extrêmement élevée pour amorce-matrice des structures d&#39;ADN de type. Ainsi, il est nécessaire d&#39;éliminer ces non-spécifiques ARNP complexes ADN-afin d&#39;effectuer la réplication de l&#39;amorce-matrice. La concentration des radio-marqués produits de l&#39;ADN en utilisant le kit de purification de PCR Qiagen est également essentiel afin d&#39;obtenir un signal assez haute radioactives qui peuvent être observées dans le gel.

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<td>Dynabeads M-270</td>
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<td>Invitrogen</td>
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<td>SapI</td>
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<td>PCR Purification Kit</td>
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direct restart of a replication fork stalled by a head-on rna polymerase

Des études in vivo suggèrent que fourches de réplication sont arrêtées en raison de rencontres avec la tête sur les complexes de transcription. Pourtant, le sort de la réplisome et l&#39;ARN polymérase (ARNP) suite à une collision frontale est inconnue. Ici, nous constatons que la E. coli stands réplisome lors d&#39;une collision avec une tête sur le complexe de transcription-, mais au lieu de s&#39;effondrer, la fourche de réplication reste très stable et reprend finalement allongement après déplacement de la ARNP partir de l&#39;ADN. Nous constatons aussi que le facteur de couplage transcription-réparation, MFD, favorise redémarrer directe de la fourche après la collision, en facilitant le déplacement de la ARNP. Ces résultats démontrent la stabilité intrinsèque de l&#39;appareil de réplication et un nouveau rôle pour la voie de réparation transcription-réplication couplée à la promotion d&#39;un bloc ARNP dernières.

Watch this Scientific Journal Video about Direct Restart of a Replication Fork Stalled by a Head-On RNA Polymerase at JoVE.com

Direct Restart of a Replication Fork Stalled by a Head-On RNA Polymerase

Le sort de la réplisome suite d&#39;une collision avec une tête sur l&#39;ARN polymérase (ARNP) est inconnu. Nous constatons que les étals réplisome lors d&#39;une collision avec un ARNP de front, mais reprend allongement après déplacement de la ARNP partir de l&#39;ADN. Mfd favorise redémarrer la réplication en facilitant le déplacement de la ARNP après la collision.

Redémarrez direct d&#39;une fourche de réplication bloquée par une polymérase ARN Head-On

In vivo studies suggest that replication forks are arrested due to encounters with head-on transcription complexes. Yet, the fate of the replisome and RNA ...

direct-restart-replication-fork-stalled-head-on-rna

Research

JoVE Journal

Biology

13.5K vues.  Rockefeller University. Le sort de la réplisome suite d'une collision avec une tête sur l'ARN polymérase (ARNP) est inconnu. Nous constatons que les étals réplisome lors d'une collision avec un ARNP de front, mais reprend allongement après déplacement de la ARNP partir de l'ADN. Mfd favorise redémarrer la réplication en facilitant le déplacement de la ARNP après la collision.

Vidéo: Direct Restart of a Replication Fork Stalled by a Head-On RNA Polymerase

Production of Replication-Defective Retrovirus by Transient Transfection of 293T cells

Our lab studies human myeloproliferative diseases induced by such oncogenes as Bcr-Abl or growth factor receptor-derived oncogenes (ZNF198-FGFR1, Bcr-PDGFR&#945;, etc.). We are able to model and study a human-like disease in our mouse model, by transplanting bone marrow cells previously infected with a retrovirus expressing the oncogene of interest. Replication-defective retrovirus encoding a human oncogene and a marker (GFP, RFP, antibiotic resistance gene, etc.) is produced by a transient transfection protocol using 293T cells, a human renal epithelial cell line transformed by the adenovirus E1A gene product. 293 cells have the unusual property of being highly transfectable by calcium phosphate (CaPO4), with up to 50-80% transfection efficiency readily attainable. Here, we co-transfect 293 cells with a retroviral vector expressing the oncogene of interest and a plasmid that expresses the gag-pol-env packaging functions, such as the single-genome packaging constructs kat or pCL, in this case the EcoPak plasmid. The initial transfection is further improved by use of chloroquine. Stocks of ecotropic virus, collected as culture supernatant 48 hrs. post-transfection, can be stored at -80&deg;C and used for infection of cell-lines in view of transformation and in vitro studies, or primary cells such as mouse bone marrow cells, that can then be used for transplant in our mouse model.

This technique demonstrates an efficient way to prepare replication-defective retroviral stocks encoding a human oncogene, and subsequently used for induction of myeloproliferative disease in the mouse model.

Production de réplication défectueuse Rétrovirus par transfection transitoire de cellules 293T

Our lab studies human myeloproliferative diseases induced by such oncogenes as Bcr-Abl or growth factor receptor-derived oncogenes (ZNF198-FGFR1, ...

Recherche

Biologie

Visualizing Single-molecule DNA Replication with Fluorescence Microscopy

We describe a simple fluorescence microscopy-based real-time method for observing DNA replication at the single-molecule level. A circular, forked DNA template is attached to a functionalized glass coverslip and replicated extensively after introduction of replication proteins and nucleotides (Figure 1). The growing product double-strand DNA (dsDNA) is extended with laminar flow and visualized by using an intercalating dye. Measuring the position of the growing DNA end in real time allows precise determination of replication rate (Figure 2). Furthermore, the length of completed DNA products reports on the processivity of replication. This experiment can be performed very easily and rapidly and requires only a fluorescence microscope with a reasonably sensitive camera.


This protocol demonstrates a simple single-molecule fluorescence microscopy technique for visualizing DNA replication by individual replisomes in real time.

Visualisation de la réplication d&#39;ADN molécule unique par microscopie à fluorescence

We describe a simple fluorescence microscopy-based real-time method for observing DNA replication at the single-molecule level. A circular, forked DNA ...

Direct Observation of Enzymes Replicating DNA Using a Single-molecule DNA Stretching Assay

We describe a method for observing real time replication of individual DNA molecules mediated by proteins of the bacteriophage replication system. Linearized &lambda; DNA is modified to have a biotin on the end of one strand, and a digoxigenin moiety on the other end of the same strand. The biotinylated end is attached to a functionalized glass coverslip and the digoxigeninated end to a small bead. The assembly of these DNA-bead tethers on the surface of a flow cell allows a laminar flow to be applied to exert a drag force on the bead. As a result, the DNA is stretched close to and parallel to the surface of the coverslip at a force that is determined by the flow rate (Figure 1). The length of the DNA is measured by monitoring the position of the bead. Length differences between single- and double-stranded DNA are utilized to obtain real-time information on the activity of the replication proteins at the fork. Measuring the position of the bead allows precise determination of the rates and processivities of DNA unwinding and polymerization (Figure 2).

We describe a method for observing real time replication of individual DNA molecules mediated by proteins of the bacteriophage replication system.

Observation directe d&#39;ADN Enzymes réplication via un ADN simple molécule Stretching Assay

We describe a method for observing real time replication of individual DNA molecules mediated by proteins of the bacteriophage replication system. ...

Visualizing RNA Localization in Xenopus Oocytes

RNA localization is a conserved mechanism of establishing cell polarity. Vg1 mRNA localizes to the vegetal pole of Xenopus laevis oocytes and acts to spatially restrict gene expression of Vg1 protein. Tight control of Vg1 distribution in this manner is required for proper germ layer specification in the developing embryo. RNA sequence elements in the 3' UTR of the mRNA, the Vg1 localization element (VLE) are required and sufficient to direct transport. To study the recognition and transport of Vg1 mRNA in vivo, we have developed an imaging technique that allows extensive analysis of trans-factor directed transport mechanisms via a simple visual readout. 
To visualize RNA localization, we synthesize fluorescently labeled VLE RNA and microinject this transcript into individual oocytes. After oocyte culture to allow transport of the injected RNA, oocytes are fixed and dehydrated prior to imaging by confocal microscopy. Visualization of mRNA localization patterns provides a readout for monitoring the complete pathway of RNA transport and for identifying roles in directing RNA transport for cis-acting elements within the transcript and trans-acting factors that bind to the VLE (Lewis et al., 2008, Messitt et al., 2008). We have extended this technique through co-localization with additional RNAs and proteins (Gagnon and Mowry, 2009, Messitt et al., 2008), and in combination with disruption of motor proteins and the cytoskeleton (Messitt et al., 2008) to probe mechanisms underlying mRNA localization.

Visualizing RNA Localization in Xenopus Oocytes

Visualization of in vivo RNA transport is accomplished by microinjection of fluorescently labeled RNA transcripts into Xenopus oocytes, followed by confocal microscopy.

Visualisation de localisation de l&#39;ARN Xenopus Les ovocytes

RNA localization is a conserved mechanism of establishing cell polarity.  Vg1 mRNA localizes to the vegetal pole of Xenopus laevis oocytes and acts to ...

Direct Delivery of MIF Morpholinos Into the Zebrafish Otocyst by Injection and Electroporation Affects Inner Ear Development

In recent years, electroporation has become a popular technique for in vivo transfection of DNA, RNA, and morpholinos into various tissues, including the eye, brain, and somites of zebrafish. The advantage of electroporation over other methods of genetic manipulation is that specific tissues can be targeted, both spatially and temporally, for the introduction of macromolecules by the application of electrical current. Here we describe the use of electroporation for transfecting mif and mif-like morpholinos into the tissues of the developing inner ear of the zebrafish. In past studies, mif morpholino injected into embryos at the 1- to 8-cell stage resulted in widespread morphological changes in the nervous system and eye, as well as the ear. By targeting the tissues of the inner ear at later stages in development, we can determine the primary effects of MIF in the developing inner ear, as opposed to secondary effects that may result from the influence of other tissues. By using phalloidin and acetylated tubulin staining to study the morphology of neurons, neuronal processes, and hair cells associated with the posterior macula, we were able to assess the efficacy of electroporation as a method for targeted transfection in the zebrafish inner ear. The otic vesicles of 24hpf embryos were injected with morpholinos and electroporated and were then compared to embryos that had received no treatment or had been only injected or electroporated. Embryos that were injected and electroporated showed a decrease in hair cell numbers, decreased innervation by the statoacoustic ganglion (SAG) and fewer SAG neurons compared with control groups. Our results showed that direct delivery of morpholinos into otocysts at later stages avoids the non-specific nervous system and neural crest effects of morpholinos delivered at the 1-8 cell stage. It also allows examination of effects that are directed to the inner ear and not secondary effects on the ear from primary effects on the brain, neural crest or periotic mesenchyme.

A method to deliver morpholinos directly into the zebrafish otocyst at 24hpf has been developed. Using microinjection of morpholinos into the lumen of otic vesicle and electroporation to effect penetration, we were able to bypass the effect of morpholinos on the brain and obtain effects specific to the inner ear.

Livraison directe de morpholinos MIF Into the otocyste Zebrafish par injection et électroporation affecte le développement oreille interne

In recent years, electroporation has become a popular technique for in vivo transfection of DNA, RNA, and morpholinos into various tissues, including the ...

In vitro Transcription and Capping of Gaussia Luciferase mRNA Followed by HeLa Cell Transfection

In vitro transcription is the synthesis of RNA transcripts by RNA polymerase from a linear DNA template containing the corresponding promoter sequence (T7, T3, SP6) and the gene to be transcribed (Figure 1A). A typical transcription reaction consists of the template DNA, RNA polymerase, ribonucleotide triphosphates, RNase inhibitor and buffer containing Mg2+ ions.
Large amounts of high quality RNA are often required for a variety of applications. Use of in vitro transcription has been reported for RNA structure and function studies such as splicing1, RNAi experiments in mammalian cells2, antisense RNA amplification by the "Eberwine method"3, microarray analysis4 and for RNA vaccine studies5. The technique can also be used for producing radiolabeled and dye labeled probes6. Warren, et al. recently reported reprogramming of human cells by transfection with in vitro transcribed capped RNA7. The T7 High Yield RNA Synthesis Kit from New England Biolabs has been designed to synthesize up to 180 &mu;g RNA per 20 &mu;l reaction. RNA of length up to 10kb has been successfully transcribed using this kit. Linearized plasmid DNA, PCR products and synthetic DNA oligonucleotides can be used as templates for transcription as long as they have the T7 promoter sequence upstream of the gene to be transcribed.
Addition of a 5' end cap structure to the RNA is an important process in eukaryotes. It is essential for RNA stability8, efficient translation9, nuclear transport10 and splicing11. The process involves addition of a 7-methylguanosine cap at the 5' triphosphate end of the RNA. RNA capping can be carried out post-transcriptionally using capping enzymes or co-transcriptionally using cap analogs. In the enzymatic method, the mRNA is capped using the Vaccinia virus capping enzyme12,13. The enzyme adds on a 7-methylguanosine cap at the 5' end of the RNA using GTP and S-adenosyl methionine as donors (cap 0 structure). Both methods yield functionally active capped RNA suitable for transfection or other applications14 such as generating viral genomic RNA for reverse-genetic systems15 and crystallographic studies of cap binding proteins such as eIF4E16.
In the method described below, the T7 High Yield RNA Synthesis Kit from NEB is used to synthesize capped and uncapped RNA transcripts of Gaussia luciferase (GLuc) and Cypridina luciferase (CLuc). A portion of the uncapped GLuc RNA is capped using the Vaccinia Capping System (NEB). A linearized plasmid containing the GLuc or CLuc gene and T7 promoter is used as the template DNA. The transcribed RNA is transfected into HeLa cells and cell culture supernatants are assayed for luciferase activity. Capped CLuc RNA is used as the internal control to normalize GLuc expression.

In vitro Transcription and Capping of Gaussia Luciferase mRNA Followed by HeLa Cell Transfection

This method describes high yield in vitro synthesis of both capped and uncapped mRNA from a linearized plasmid containing the Gaussia luciferase (GLuc) gene. The RNA is purified and a fraction of the uncapped RNA is enzymatically capped using the Vaccinia virus capping enzyme. In the final step, the mRNA is transfected into HeLa cells and cell culture supernatants are assayed for luciferase activity.

Transcription in vitro et plafonnement de l&#39;ARNm de luciférase Gaussia Suivie par la transfection de cellules HeLa

In vitro transcription is the synthesis of RNA transcripts by RNA polymerase from a linear DNA template containing the corresponding promoter sequence ...

RNA In situ Hybridization in Whole Mount Embryos and Cell Histology Adapted for Marine Elasmobranchs

Marine elasmobranchs are valued animal models for biomedical and genomic studies as they are the most primitive vertebrates to have adaptive immunity and have unique mechanisms for osmoregulation 1-3. As the most primitive living jawed-vertebrates with paired appendages, elasmobranchs are an evolutionarily important model, especially for studies in evolution and development. Marine elasmobranchs have also been used to study aquatic toxicology and stress physiology in relationship to climate change 4. Thus, development and adaptation of methodologies is needed to facilitate and expand the use of these primitive vertebrates to multiple biological disciplines. Here I present the successful adaptation of RNA whole mount in situ hybridization and histological techniques to study gene expression and cell histology in elasmobranchs.
Monitoring gene expression is a hallmark tool of developmental biologists, and is widely used to investigate developmental processes 5. RNA whole mount in situ hybridization allows for the visualization and localization of specific gene transcripts in tissues of the developing embryo. The expression pattern of a gene's message can provide insight into what developmental processes and cell fate decisions a gene may control. By comparing the expression pattern of a gene at different developmental stages, insight can be gained into how the role of a gene changes during development.
While whole mount in situ's provides a means to localize gene expression to tissue, histological techniques allow for the identification of differentiated cell types and tissues. Histological stains have varied functions. General stains are used to highlight cell morphology, for example hematoxylin and eosin for general staining of nuclei and cytoplasm, respectively. Other stains can highlight specific cell types. For example, the alcian blue stain reported in this paper is a widely used cationic stain to identify mucosaccharides. Staining of the digestive tract with alcian blue can identify the distribution of goblet cells that produce mucosaccharides. Variations in mucosaccharide constituents on short peptides distinguish goblet cells by function within the digestive tract 6. By using RNA whole mount in situ's and histochemical methods concurrently, cell fate decisions can be linked to gene-specific expression.
Although RNA in situ's and histochemistry are widely used by researchers, their adaptation and use in marine elasmobranchs have met limited and varied success. Here I present protocols developed for elasmobranchs and used on a regular basis in my laboratory. Although further modification of the RNA in situ's hybridization method may be needed to adapt to different species, the protocols described here provide a strong starting point for researchers wanting to adapt the use of marine elasmobranchs to their scientific inquiries.

RNA In situ Hybridization in Whole Mount Embryos and Cell Histology Adapted for Marine Elasmobranchs

By combining methods for RNA whole mount in situ hybridization and histology, gene expression can be linked with cell fate decisions in the developing embryo. These methods have been adapted to marine elasmobranchs and facilitate the use of these animals as model organisms for biomedical, toxicology and comparative studies.

ARN<em&gt; In situ</emHybridation&gt; en embryons entiers de montage et histologie Adapté pour les élasmobranches marins

Marine  elasmobranchs are valued animal models for biomedical and genomic studies as  they are the most primitive vertebrates to have adaptive immunity ...

RNA Isolation from Mouse Pancreas: A Ribonuclease-rich Tissue

Isolation of high-quality RNA from ribonuclease-rich tissue such as mouse pancreas presents a challenge. As a primary function of the pancreas is to aid in digestion, mouse pancreas may contain as much a 75 mg of ribonuclease. We report modifications of standard phenol/guanidine thiocyanate lysis reagent protocols to isolate RNA from mouse pancreas. Guanidine thiocyanate is a strong protein denaturant and will effectively disrupt the activity of ribonuclease under most conditions. However, critical modifications to standard protocols are necessary to successfully isolate RNA from ribonuclease-rich tissues. Key steps include a high lysis reagent to tissue ratio, removal of undigested tissue prior to phase separation and inclusion of a ribonuclease inhibitor to the RNA solution. Using these and other modifications, we routinely isolate RNA with RNA Integrity Number (RIN) greater than 7. The isolated RNA is of suitable quality for routine gene expression analysis. Adaptation of this protocol to isolate RNA from ribonuclease rich tissues besides the pancreas should be readily achievable.

We report a procedure to isolate RNA with high integrity from the ribonuclease rich mouse pancreas.

Isolement de l&#39;ARN de souris Pancréas: un tissu de Ribonuclease riche

Isolation of high-quality RNA from ribonuclease-rich tissue such as mouse pancreas presents a challenge.  As a primary function of the pancreas is to aid ...

Development of a Quantitative Recombinase Polymerase Amplification Assay with an Internal Positive Control

It was recently demonstrated that recombinase polymerase amplification (RPA), an isothermal amplification platform for pathogen detection, may be used to quantify DNA sample concentration using a standard curve. In this manuscript, a detailed protocol for developing and implementing a real-time quantitative recombinase polymerase amplification assay (qRPA assay) is provided. Using HIV-1 DNA quantification as an example, the assembly of real-time RPA reactions, the design of an internal positive control (IPC) sequence, and co-amplification of the IPC and target of interest are all described. Instructions and data processing scripts for the construction of a standard curve using data from multiple experiments are provided, which may be used to predict the concentration of unknown samples or assess the performance of the assay. Finally, an alternative method for collecting real-time fluorescence data with a microscope and a stage heater as a step towards developing a point-of-care qRPA assay is described. The protocol and scripts provided may be used for the development of a qRPA assay for any DNA target of interest.

Provided is a protocol for developing a real-time recombinase polymerase amplification assay to quantify initial concentration of DNA samples using either a thermal cycler or a microscope and stage heater. Also described is the development of an internal positive control. Scripts are provided for processing raw real-time fluorescence data.

Développement d&#39;un Quantitative recombinase polymérase L&#39;amplification Assay avec un contrôle positif interne

It was recently demonstrated that recombinase polymerase amplification (RPA), an isothermal amplification platform for pathogen detection, may be used to ...

Inducing a Site Specific Replication Blockage in E. coli Using a Fluorescent Repressor Operator System

Obstacles present on DNA, including tightly-bound proteins and various lesions, can severely inhibit the progression of the cell&#39;s replication machinery. The stalling of a replisome can lead to its dissociation from the chromosome, either in part or its entirety, leading to the collapse of the replication fork. The recovery from this collapse is a necessity for the cell to accurately complete chromosomal duplication and subsequently divide. Therefore, when the collapse occurs, the cell has evolved diverse mechanisms that take place to restore the DNA fork and allow replication to be completed with high fidelity. Previously, these replication repair pathways in bacteria have been studied using UV damage, which has the disadvantage of not being localized to a known site. This manuscript describes a system utilizing a Fluorescence Repressor Operator System (FROS) to create a site-specific protein block that can induce the stalling and collapse of replication forks in Escherichia coli. Protocols detail how the status of replication can be visualized in single living cells using fluorescence microscopy and DNA replication intermediates can be analyzed by 2-dimensional agarose gel electrophoresis. Temperature sensitive mutants of replisome components (e.g. DnaBts) can be incorporated into the system to induce a synchronous collapse of the replication forks. Furthermore, the roles of the recombination proteins and helicases that are involved in these processes can be studied using genetic knockouts within this system.

Inducing a Site Specific Replication Blockage in E. coli Using a Fluorescent Repressor Operator System

We describe here a system utilizing a site-specific, reversible in vivo protein block to stall and collapse replication forks in Escherichia coli. The establishment of the replication block is evaluated by fluorescence microscopy and neutral-neutral 2-dimensional agarose gel electrophoresis is used to visualize replication intermediates.