Ce travail a été soutenu par le NIH subventions GM45916 et GM66834 à JCH et une bourse de la Fondation internationale pour le syndrome de Rett AK Ce travail a également été soutenu par des subventions du NIH GM088409 et Howard Hughes Medical Institute contributions à KL

<ol>
	<li>Luger, K., Mader, A., Richmond, R., Crystal Sargent, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=structure+of+the+nucleosome+core+particle+at+2.8+A+resolution.">structure of the nucleosome core particle at 2.8 A resolution.</a> Nature. 7, (1997).</li><li>Hansen, J. 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Les auteurs n&#39;ont aucun conflit d&#39;intérêt.

Modèle tableaux de nucléosomiques sont un outil très utile pour l&#39;étude in vitro de la structure et la fonction de la chromatine. Par exemple, ils ont été largement utilisés pour étudier le mécanisme de la condensation de la chromatine de la fibre dans une solution de 30 à 34, et a permis d&#39;obtenir une structure aux rayons X d&#39;un Tetranucleosome 35. Plus récemment, ils se sont avérés utiles à déchiffrer les effets structurels de variantes noyau d&#39;histone spé...

Génomes eucaryotes n&#39;existent pas d&#39;ADN nu, mais sont compactés et organisées par des protéines liées. Ces complexes d&#39;ADN et de protéines sont connues comme la chromatine. L&#39;unité de répétition de base de la chromatine est le nucléosome. Un nucléosome est constitué d&#39;histone octamère et 146 paires de bases d&#39;ADN enroulé autour de l&#39;octamère d&#39;histone environ 1,6 fois 1. L&#39;octamère d&#39;histones est composé de deux exemplaires de chacun des histones H2A le fondamentales, H2B, H3, et H4. Octamères histones fondamentales qui sont répétitive espacés le long d&#39;une molécule d&#39;ADN sont appelés tableaux nucléosomiques. La structure étendue de tableaux nucléosomiques a été désigné comme la fibre de 10 nm ou les «perles sur un fil&quot; structure et est présent in vitro dans des conditions de faible teneur en sel 2. La fibre de 10 nm est capable de se condenser dans les structures d&#39;ordre supérieur par le compactage intra-groupe et / ou inter-réseau oligomérisation 2. Ces structures d&#39;ordre supérieur peuvent être induites en présence de sels,ou qui peut être influencé par la liaison des protéines de la chromatine à l&#39;architecture du réseau nucléosomale 3,4. Niveaux de compaction de la chromatine sont inversement corrélés avec un taux de transcription dans 5,6 vivo. Les dernières recherches soulignent l&#39;importance de l&#39;organisation structurale des génomes dans des procédés tels que la différenciation, le développement du cancer et d&#39;autres 7,8. L&#39;utilisation de tableaux nucléosomiques pour étudier la structure et la fonction de la chromatine s&#39;est généralisée. Nous décrivons ici une méthode pour l&#39;assemblage de tableaux de nucléosomiques histones recombinantes et d&#39;ADN de positionnement des nucléosomes. Utilisation de l&#39;ADN recombinant avec des répétitions en tandem de séquences de positionnement des nucléosomes permet la reconstitution des tableaux qui contiennent des nucléosomes régulièrement espacés. Deux des séquences de positionnement plus populaires sont la séquence du gène d&#39;ARNr 5S et le &quot;601&quot; 9,10 de séquence. La séquence 601 a été obtenue à partir d&#39;expériences et la mortalité SELEXe positionne fortement nucléosomes que la séquence 5S 11. Par conséquent, la longueur de liaison de l&#39;ADN des 601 matrices est plus homogène. ADN de positionnement des nucléosomes répétée en tandem est obtenu par filtration sur gel 4,12. Histones recombinantes sont purifiées à partir de E. coli dans des conditions de dénaturation 13. L&#39;utilisation d&#39;histones recombinantes permet de contrôler avec soin la composition de l&#39;histone des matrices nucléosomiques. Par exemple, le noyau histones portant des mutations spécifiques 14 ou modifications post-traductionnelles 15,16 peut être substitué pour histones de type sauvage. expériences de vitesse de sédimentation de surveiller le taux de sédimentation des macromolécules en solution sous une force centrifuge appliquée 17. Cela donne des informations sur la taille et la forme des macromolécules dans un échantillon. expériences de vitesse de sédimentation sont donc un outil approprié pour l&#39;étude des changements d&#39;état de la solution dans la fibre de chromatinela structure due à la condensation de la chromatine 18. Surtout, il est d&#39;abord nécessaire d&#39;utiliser des expériences de vitesse de sédimentation comme une étape de contrôle de la qualité dans nucléosomique tableau reconstitution. Si l&#39;ADN et les nucléosomes sont pas combinées à un rapport molaire approprié, les tableaux peuvent être sous-ou sur-saturé avec histones. Ainsi, les informations obtenues à partir d&#39;expériences de vitesse de sédimentation est utilisé pour veiller à ce que l&#39;ADN est correctement saturé de nucléosomes. Il est important d&#39;utiliser d&#39;autres méthodes pour estimer la saturation de l&#39;ADN avec des nucléosomes, en particulier si l&#39;on travaille avec une matrice d&#39;ADN non caractérisée. Par conséquent, nous décrivons également un procédé pour l&#39;analyse de réseaux nucléosomales utilisant la microscopie à force atomique (AFM). L&#39;AFM est une technique puissante qui permet la visualisation des effets d&#39;un certain nombre de paramètres, tels que le niveau de saturation, l&#39;effet de la présence de variants d&#39;histones ou les effets de MgCl 2 19,20. AFM a également été applied pour étudier la dynamique des nucléosomes utilisant laps de temps imagerie 21. In vitro assemblés nucléosomiques tableaux 12-mer sont particulièrement favorables à des études de l&#39;AFM, car ils appartiennent à la gamme de la bonne taille pour l&#39;imagerie AFM 22. Dans la présente étude, nous avons utilisé l&#39;AFM de tableaux nucléosomiques comme un contrôle de la qualité ainsi que d&#39;un moyen d&#39;affirmer les données de l&#39;ASC (&quot;voir c&#39;est croire&quot;). En plus de la visualisation simple, l&#39;AFM permet de mesurer des profils de hauteur d&#39;échantillons comme une métrique supplémentaire.

<table border="1">
 <tbody>
 <tr>
 <td>Name of Reagent/Material</td>
 <td>Company</td>
 <td>Catalog Number</td>
 <td>Comments</td>
 </tr>
 <tr>
 <td>(3-Aminopropyl)triethoxysilane</td>
 <td>Sigma-Aldrich </td>
 <td>A3648-100ML</td>
 <td></td>
 </tr>
 <tr>
 <td>6-8 kDa MWCO Dialysis Tubing</td>
 <td>Fisher</td>
 <td>21-152-5</td>
 <td></td>
 </tr>
 <tr>
 <td>HiLoad Superdex 200 16/60 Column</td>
 <td>GE</td>
 <td>17-1069-01</td>
 <td></td>
 </tr>
 <tr>
 <td>Vivaspin 50 kDa MWCO Centrifugal Concentrator</td>
 <td>Sartorius</td>
 <td>VS2031</td>
 <td></td>
 </tr>
 <tr>
 <td>12-14 kDa MWCO Dialysis Tubing</td>
 <td>Fisher</td>
 <td>08-667A</td>
 <td></td>
 </tr>
 <tr>
 <td>Illustra Sephacryl S-1000 Superfine</td>
 <td>GE</td>
 <td>17-0476-01 </td>
 <td></td>
 </tr>
 <tr>
 <td>XL-A/I Analytical Ultracentrifuge</td>
 <td>Beckman-Coulter</td>
 <td></td>
 <td></td>
 </tr>
 </tbody>
</table>

assembly of nucleosomal arrays from recombinant core histones and nucleosome positioning dna

Octamères histones fondamentales qui sont répétitive espacés le long d&#39;une molécule d&#39;ADN sont appelés tableaux nucléosomiques. Tableaux nucléosomes sont obtenus dans une des deux façons suivantes: purification à partir de sources in vivo ou in vitro de reconstitution histones fondamentales recombinants et de l&#39;ADN répétées en tandem de positionnement des nucléosomes. Cette dernière méthode a l&#39;avantage de permettre à l&#39;ensemble d&#39;un uniforme plus de composition et tableau nucléosomique positionné précisément. expériences de vitesse de sédimentation en ultracentrifugation analytique des informations d&#39;élasticité d&#39;environ la taille et la forme des macromolécules en analysant la vitesse à laquelle ils migrent à travers la solution sous la force centrifuge. Cette technique, en même temps que la microscopie à force atomique, peut être utilisé pour le contrôle de qualité, en veillant à ce que la majorité des matrices d&#39;ADN sont saturés avec des nucléosomes après reconstitution. Nous décrivons ici les protocoles nécessaires pour reconstituer quelques milligrammes de longueur et de composition dtableaux nucléosomiques efined appropriés pour les études biochimiques et biophysiques de la structure et la fonction de la chromatine.

Pour illustrer le protocole nous avons reconstitué à partir de matrices histones nucléosomiques recombinants de base de Xenopus et de l&#39;ADN comprenant des 12 séquences répétées en tandem de 207 bp de la séquence de positionnement 601 (601 207 x 12). Nous avons d&#39;abord assemblé octamers natives de histones fondamentales lyophilisées et ensuite purifié par des octamères FPLC en utilisant une colonne S200 (Figure 1A). Grands complexes élues tôt de la colonne S200. ...

Watch this Scientific Journal Video about Assembly of Nucleosomal Arrays from Recombinant Core Histones and Nucleosome Positioning DNA at JoVE.com

Assembly of Nucleosomal Arrays from Recombinant Core Histones and Nucleosome Positioning DNA

Une méthode est présentée pour la reconstitution du modèle nucléosomiques tableaux de histones recombinantes et d&#39;ADN répétées en tandem de positionnement des nucléosomes. Nous décrivons également comment les expériences de la vitesse de sédimentation en ultracentrifugation analytique, et la microscopie à force atomique (AFM) sont utilisés pour mesurer l&#39;ampleur de nucléosomique saturation de tableau après reconstitution.

Assemblée des nucléosomiques tableaux de recombinants base histones et nucléosomes ADN positionnement

Core histone octamers that are repetitively spaced along a DNA molecule  are called nucleosomal arrays. Nucleosomal arrays are obtained in one of two  ...

assembly-nucleosomal-arrays-from-recombinant-core-histones-nucleosome

Research

Education

JoVE Core

JoVE Journal

Anatomy and Physiology

Biology

Unit 1

Cells and their Components

SCIENTISTS IN ACTION

22.4K vues.  Colorado State University.  Une méthode est présentée pour la reconstitution du modèle nucléosomiques tableaux de histones recombinantes et d'ADN répétées en tandem de positionnement des nucléosomes. Nous décrivons également comment les expériences de la vitesse de sédimentation en ultracentrifugation analytique, et la microscopie à force atomique (AFM) sont utilisés pour mesurer l'ampleur de nucléosomique saturation de tableau après reconstitution. 

Vidéo: Assembly of Nucleosomal Arrays from Recombinant Core Histones and Nucleosome Positioning DNA

Isolation of Genomic DNA from Mouse Tails

Isolement de l&#39;ADN génomique de souris Tails

Recherche

Biologie

Combining QD-FRET and Microfluidics to Monitor DNA Nanocomplex Self-Assembly in Real-Time

Advances in genomics continue to fuel the development of therapeutics that can target pathogenesis at the cellular and molecular level. Typically functional inside the cell, nucleic acid-based therapeutics require an efficient intracellular delivery system. One widely adopted approach is to complex DNA with a gene carrier to form nanocomplexes via electrostatic self-assembly, facilitating cellular uptake of DNA while protecting it against degradation. The challenge lies in the rational design of efficient gene carriers, since premature dissociation or overly stable binding would be detrimental to the cellular uptake and therapeutic efficacy. Nanocomplexes synthesized by bulk mixing showed a diverse range of intracellular unpacking and trafficking behavior, which was attributed to the heterogeneity in size and stability of nanocomplexes. Such heterogeneity hinders the accurate assessment of the self-assembly kinetics and adds to the difficulty in correlating their physical properties to transfection efficiencies or bioactivities. We present a novel convergence of nanophotonics (i.e. QD-FRET) and microfluidics to characterize the real-time kinetics of the nanocomplex self-assembly under laminar flow. QD-FRET provides a highly sensitive indication of the onset of molecular interactions and quantitative measure throughout the synthesis process, whereas microfluidics offers a well-controlled microenvironment to spatially analyze the process with high temporal resolution (~milliseconds). For the model system of polymeric nanocomplexes, two distinct stages in the self-assembly process were captured by this analytic platform. The kinetic aspect of the self-assembly process obtained at the microscale would be particularly valuable for microreactor-based reactions which are relevant to many micro- and nano-scale applications. Further, nanocomplexes may be customized through proper design of microfludic devices, and the resulting QD-FRET polymeric DNA nanocomplexes could be readily applied for establishing structure-function relationships. 

We present a novel and powerful integration of nanophotonics (QD-FRET) and microfluidics to investigate the formation of polyelectrolyte polyplexes, which is expected to provide better control and synthesis of uniform and customizable polyplexes for future nucleic acid-based therapeutics.

Combinant QD-FRET et la microfluidique pour surveiller Nanocomplex ADN auto-assemblage en temps réel

Advances in genomics continue to fuel the development of therapeutics that can target pathogenesis at the cellular and molecular level. Typically ...

Assembly, Loading, and Alignment of an Analytical Ultracentrifuge Sample Cell

The analytical ultracentrifuge (AUC) is a powerful biophysical tool that allows us to record macromolecular sedimentation profiles during high speed centrifugation. When properly planned and executed, an AUC sedimentation velocity or sedimentation equilibrium experiment can reveal a great deal about a protein in regards to size and shape, sample purity, sedimentation coefficient, oligomerization states and protein-protein interactions.
This technique, however, requires a rigorous level of technical attention. Sample cells hold a sectored center piece sandwiched between two window assemblies. They are sealed with a torque pressure of around 120-140 in/lbs. Reference buffer and sample are loaded into the centerpiece sectors and then after sealing, the cells are precisely aligned into a titanium rotor so that the optical detection systems scan both sample and reference buffer in the same radial path midline through each centerpiece sector while rotating at speeds of up to 60, 000 rpm and under very high vacuum
Not only is proper sample cell assembly critical, sample cell components are very expensive and must be properly cared for to ensure they are in optimum working condition in order to avoid leaks and breakage during experiments. Handle windows carefully, for even the slightest crack or scratch can lead to breakage in the centrifuge. The contact between centerpiece and windows must be as tight as possible; i.e. no Newton s rings should be visible after torque pressure is applied. Dust, lint, scratches and oils on either the windows or the centerpiece all compromise this contact and can very easily lead to leaking of solutions from one sector to another or leaking out of the centerpiece all together. Not only are precious samples lost, leaking of solutions during an experiment will cause an imbalance of pressure in the cell that often leads to broken windows and centerpieces. In addition, plug gaskets and housing plugs must be securely in place to avoid solutions being pulled out of the centerpiece sector through the loading holes by the high vacuum in the centrifuge chamber. Window liners and gaskets must be free of breaks and cracks that could cause movement resulting in broken windows.
This video will demonstrate our procedures of sample cell assembly, torque, loading and rotor alignment to help minimize component damage, solution leaking and breakage during the perfect AUC experiment.

The analytical ultracentrifuge (AUC) sample cell holds sample and reference buffer and during experiments and is exposed to high vacuum and rotor speeds up to 60,000 rpm. This video will demonstrate the rigorous attention to detail necessary for assembly, loading and alignment of this very important component of an AUC experiment.

Assemblée, de chargement, et l&#39;alignement d&#39;une cellule échantillon ultracentrifugation analytique

The analytical ultracentrifuge (AUC) is a powerful biophysical tool that allows us to record macromolecular sedimentation profiles during high speed ...

Extraction of High Molecular Weight Genomic DNA from Soils and Sediments

The soil microbiome is a vast and relatively unexplored reservoir of genomic diversity and metabolic innovation that is intimately associated with nutrient and energy flow within terrestrial ecosystems. Cultivation-independent environmental genomic, also known as metagenomic, approaches promise unprecedented access to this genetic information with respect to pathway reconstruction and functional screening for high value therapeutic and biomass conversion processes. However, the soil microbiome still remains a challenge largely due to the difficulty in obtaining high molecular weight DNA of sufficient quality for large insert library production. Here we introduce a protocol for extracting high molecular weight, microbial community genomic DNA from soils and sediments. The quality of isolated genomic DNA is ideal for constructing large insert environmental genomic libraries for downstream sequencing and screening applications. 
The procedure starts with cell lysis. Cell walls and membranes of microbes are lysed by both mechanical (grinding) and chemical forces (&beta;-mercaptoethanol). Genomic DNA is then isolated using extraction buffer, chloroform-isoamyl alcohol and isopropyl alcohol. The buffers employed for the lysis and extraction steps include guanidine isothiocyanate and hexadecyltrimethylammonium bromide (CTAB) to preserve the integrity of the high molecular weight genomic DNA. Depending on your downstream application, the isolated genomic DNA can be further purified using cesium chloride (CsCl) gradient ultracentrifugation, which reduces impurities including humic acids. The first procedure, extraction, takes approximately 8 hours, excluding DNA quantification step. The CsCl gradient ultracentrifugation, is a two days process. During the entire procedure, genomic DNA should be treated gently to prevent shearing, avoid severe vortexing, and repetitive harsh pipetting.

A methodology to isolate high molecular weight and high quality genomic DNA from soil microbial community is described.

Extraction d&#39;ADN de haut poids moléculaire génomique par les sols et sédiments

The soil microbiome is a vast and relatively unexplored reservoir of genomic diversity and metabolic innovation that is intimately associated with ...

Selective Capture of 5-hydroxymethylcytosine from Genomic DNA

5-methylcytosine (5-mC) constitutes ~2-8% of the total cytosines in human genomic DNA and impacts a broad range of biological functions, including gene expression, maintenance of genome integrity, parental imprinting, X-chromosome inactivation, regulation of development, aging, and cancer1. Recently, the presence of an oxidized 5-mC, 5-hydroxymethylcytosine (5-hmC), was discovered in mammalian cells, in particular in embryonic stem (ES) cells and neuronal cells2-4. 5-hmC is generated by oxidation of 5-mC catalyzed by TET family iron (II)/&alpha;-ketoglutarate-dependent dioxygenases2, 3. 5-hmC is proposed to be involved in the maintenance of embryonic stem (mES) cell, normal hematopoiesis and malignancies, and zygote development2, 5-10. To better understand the function of 5-hmC, a reliable and straightforward sequencing system is essential. Traditional bisulfite sequencing cannot distinguish 5-hmC from 5-mC11. To unravel the biology of 5-hmC, we have developed a highly efficient and selective chemical approach to label and capture 5-hmC, taking advantage of a bacteriophage enzyme that adds a glucose moiety to 5-hmC specifically12.
Here we describe a straightforward two-step procedure for selective chemical labeling of 5-hmC. In the first labeling step, 5-hmC in genomic DNA is labeled with a 6-azide-glucose catalyzed by &beta;-GT, a glucosyltransferase from T4 bacteriophage, in a way that transfers the 6-azide-glucose to 5-hmC from the modified cofactor, UDP-6-N3-Glc (6-N3UDPG). In the second step, biotinylation, a disulfide biotin linker is attached to the azide group by click chemistry. Both steps are highly specific and efficient, leading to complete labeling regardless of the abundance of 5-hmC in genomic regions and giving extremely low background. Following biotinylation of 5-hmC, the 5-hmC-containing DNA fragments are then selectively captured using streptavidin beads in a density-independent manner. The resulting 5-hmC-enriched DNA fragments could be used for downstream analyses, including next-generation sequencing.
Our selective labeling and capture protocol confers high sensitivity, applicable to any source of genomic DNA with variable/diverse 5-hmC abundances. Although the main purpose of this protocol is its downstream application (i.e., next-generation sequencing to map out the 5-hmC distribution in genome), it is compatible with single-molecule, real-time SMRT (DNA) sequencing, which is capable of delivering single-base resolution sequencing of 5-hmC.

Described is a two-step labeling process using &beta;-glucosyltransferase (&beta;-GT) to transfer an azide-glucose to 5-hmC, followed by click chemistry to transfer a biotin linker for easy and density-independent enrichment. This efficient and specific labeling method enables enrichment of 5-hmC with extremely low background and high-throughput epigenomic mapping via next-generation sequencing.

Capture d&#39;sélectif de 5-hydroxyméthylcytosine partir de l&#39;ADN génomique

5-methylcytosine (5-mC) constitutes ~2-8% of the total cytosines in human genomic DNA and impacts a broad range of biological functions, including gene ...

Design and Use of Multiplexed Chemostat Arrays

Chemostats are continuous culture systems in which cells are grown in a tightly controlled, chemically constant environment where culture density is constrained by limiting specific nutrients.1,2 Data from chemostats are highly reproducible for the measurement of quantitative phenotypes as they provide a constant growth rate and environment at steady state. For these reasons, chemostats have become useful tools for fine-scale characterization of physiology through analysis of gene expression3-6 and other characteristics of cultures at steady-state equilibrium.7 Long-term experiments in chemostats can highlight specific trajectories that microbial populations adopt during adaptive evolution in a controlled environment. In fact, chemostats have been used for experimental evolution since their invention.8 A common result in evolution experiments is for each biological replicate to acquire a unique repertoire of mutations.9-13 This diversity suggests that there is much left to be discovered by performing evolution experiments with far greater throughput. 
We present here the design and operation of a relatively simple, low cost array of miniature chemostats&mdash;or ministats&mdash;and validate their use in determination of physiology and in evolution experiments with yeast. This approach entails growth of tens of chemostats run off a single multiplexed peristaltic pump. The cultures are maintained at a 20 ml working volume, which is practical for a variety of applications. It is our hope that increasing throughput, decreasing expense, and providing detailed building and operation instructions may also motivate research and industrial application of this design as a general platform for functionally characterizing large numbers of strains, species, and growth parameters, as well as genetic or drug libraries.

We developed and validated a small-footprint array of miniature chemostats built from readily available parts for low cost. Physiological and experimental evolution results were similar to larger volume chemostats. The ministat array provides a compact, inexpensive, and accessible platform for traditional chemostat experiments, functional genomics, and chemical screening applications.

Conception et utilisation des tableaux chémostat multiplexés

Chemostats are continuous culture systems in which cells are grown in a tightly controlled, chemically constant environment where culture density is ...

Efficient Production and Purification of Recombinant Murine Kindlin-3 from Insect Cells for Biophysical Studies

Kindlins are essential coactivators, with talin, of the cell surface receptors&#160;integrins and also participate in integrin outside-in signalling, and the control of gene transcription in the cell nucleus. The kindlins are ~75 kDa multidomain proteins and bind to an NPxY motif and upstream T/S cluster of the integrin &#946;-subunit cytoplasmic tail. The hematopoietically-important kindlin isoform, kindlin-3, is critical for platelet aggregation during thrombus formation, leukocyte rolling in response to infection and inflammation and osteoclast podocyte formation in bone resorption. Kindlin-3&#39;s role in these processes has resulted in extensive cellular and physiological studies. However, there is a need for an efficient method of acquiring high quality milligram quantities of the protein for further studies. We have developed a protocol, here described, for the efficient expression and purification of recombinant murine kindlin-3 by use of a baculovirus-driven expression system in Sf9 cells yielding sufficient amounts of high purity full-length protein to allow its biophysical characterization. The same approach could be taken in the study of the other mammalian kindlin isoforms.

Kindlins are fundamental to cell adhesion through integrins but studies of them have been hampered by the difficulty encountered in expressing them recombinantly in bacterial hosts. We describe here methods for their efficient production in baculovirus-infected insect cells.

La production efficace et purification de recombinant murin Kindlin-3 à partir de cellules d&#39;insectes pour les études biophysiques

Kindlins are essential coactivators, with talin, of the cell surface receptors&#160;integrins and also participate in integrin outside-in signalling, and ...

Real-time Imaging of Single Engineered RNA Transcripts in Living Cells Using Ratiometric Bimolecular Beacons

The growing realization that both the temporal and spatial regulation of gene expression can have important consequences on cell function has led to the development of diverse techniques to visualize individual RNA transcripts in single living cells. One promising technique that has recently been described utilizes an oligonucleotide-based optical probe, ratiometric bimolecular beacon (RBMB), to detect RNA transcripts that were engineered to contain at least four tandem repeats of the RBMB target sequence in the 3&#8217;-untranslated region. RBMBs are specifically designed to emit a bright fluorescent signal upon hybridization to complementary RNA, but otherwise remain quenched. The use of a synthetic probe in this approach allows photostable, red-shifted, and highly emissive organic dyes to be used for imaging. Binding of multiple RBMBs to the engineered RNA transcripts results in discrete fluorescence spots when viewed under a wide-field fluorescent microscope. Consequently, the movement of individual RNA transcripts can be readily visualized in real-time by taking a time series of fluorescent images. Here we describe the preparation and purification of RBMBs, delivery into cells by microporation and live-cell imaging of single RNA transcripts.

Ratiometric bimolecular beacons (RBMBs) can be used to image single engineered RNA transcripts in living cells. Here, we describe the preparation and purification of RBMBs, delivery of RBMBs into cells by microporation and fluorescent imaging of single RNA transcripts in real-time.

Imagerie en temps réel de l&#39;unité d&#39;ingénierie des ARN transcrits dans des cellules vivantes utilisant Ratiométrique bimoléculaires Beacons

The growing realization that both the temporal and spatial regulation of gene expression can have important consequences on cell function has led to the ...

Non-chromatographic Purification of Recombinant Elastin-like Polypeptides and their Fusions with Peptides and Proteins from Escherichia coli

Elastin-like polypeptides are repetitive biopolymers that exhibit a lower critical solution temperature phase transition behavior, existing as soluble unimers below a characteristic transition temperature and aggregating into micron-scale coacervates above their transition temperature. The design of elastin-like polypeptides at the genetic level permits precise control of their sequence and length, which dictates their thermal properties. Elastin-like polypeptides are used in a variety of applications including biosensing, tissue engineering, and drug delivery, where the transition temperature and biopolymer architecture of the ELP can be tuned for the specific application of interest. Furthermore, the lower critical solution temperature phase transition behavior of elastin-like polypeptides allows their purification by their thermal response, such that their selective coacervation and resolubilization allows the removal of both soluble and insoluble contaminants following expression in Escherichia coli. This approach can be used for the purification of elastin-like polypeptides alone or as a purification tool for peptide or protein fusions where recombinant peptides or proteins genetically appended to elastin-like polypeptide tags can be purified without chromatography. This protocol describes the purification of elastin-like polypeptides and their peptide or protein fusions and discusses basic characterization techniques to assess the thermal behavior of pure elastin-like polypeptide products.

Non-chromatographic Purification of Recombinant Elastin-like Polypeptides and their Fusions with Peptides and Proteins from Escherichia coli

Elastin-like polypeptides are stimulus-responsive biopolymers with applications ranging from recombinant protein purification to drug delivery. This protocol describes the purification and characterization of elastin-like polypeptides and their peptide or protein fusions from Escherichia coli using their lower critical solution temperature phase transition behavior as a simple alternative to chromatography.

Purification non chromatographique de polypeptides recombinants élastine et de leurs fusions avec des peptides et des protéines<em&gt; Escherichia coli</em

Elastin-like polypeptides are repetitive biopolymers that exhibit a lower critical solution temperature phase transition behavior, existing as soluble ...

Amplification, Next-generation Sequencing, and Genomic DNA Mapping of Retroviral Integration Sites

Retroviruses exhibit signature integration preferences on both the local and global scales. Here, we present a detailed protocol for (1) generation of diverse libraries of retroviral integration sites using ligation-mediated PCR (LM-PCR) amplification and next-generation sequencing (NGS), (2) mapping the genomic location of each virus-host junction using BEDTools, and (3) analyzing the data for statistical relevance. Genomic DNA extracted from infected cells is fragmented by digestion with restriction enzymes or by sonication. After suitable DNA end-repair, double-stranded linkers are ligated onto the DNA ends, and semi-nested PCR is conducted using primers complementary to both the long terminal repeat (LTR) end of the virus and the ligated linker DNA. The PCR primers carry sequences required for DNA clustering during NGS, negating the requirement for separate adapter ligation. Quality control (QC) is conducted to assess DNA fragment size distribution and adapter DNA incorporation prior to NGS. Sequence output files are filtered for LTR-containing reads, and the sequences defining the LTR and the linker are cropped away. Trimmed host cell sequences are mapped to a reference genome using BLAT and are filtered for minimally 97% identity to a unique point in the reference genome. Unique integration sites are scrutinized for adjacent nucleotide (nt) sequence and distribution relative to various genomic features. Using this protocol, integration site libraries of high complexity can be constructed from genomic DNA in three days. The entire protocol that encompasses exogenous viral infection of susceptible tissue culture cells to integration site analysis can therefore be conducted in approximately one to two weeks. Recent applications of this technology pertain to longitudinal analysis of integration sites from HIV-infected patients.

We describe a protocol for amplifying retroviral integration sites from the genomic DNA of infected cells, sequencing the amplified virus-host junctions, and then mapping these sequences to a reference genome. We also describe techniques to quantify the distribution of integration sites relative to various genomic annotations using BEDTools.

Amplification, Séquençage de nouvelle génération, et l&#39;ADN génomique Cartographie des rétrovirales Sites d&#39;intégration

Retroviruses exhibit signature integration preferences on both the local and global scales. Here, we present a detailed protocol for (1) generation of ...