Induktion und Überwachung von chronisch-rezidivierenden EAE Die Emulsion ist eine 1:1-Mischung von Antigen in wässriger Lösung hinzugefügt ergänzt komplettem Freund-Adjuvans. Es ist sehr wichtig, um das Antigen an das Adjuvans und nicht umgekehrt hinzuzufügen! Es gibt eine Menge von Abfällen, wenn Herstellung und Injektion einer Emulsion. Immer vorzubereiten 1,5 oder 2-mal mehr als das, was Sie brauchen. Verwenden Sie autoklaviert Mörtel, Pistill, Glasspritzen und Brücken. Alle Kunststoffe sollten steril sein. Das Präparat kann in regelmäßigen Labortisch durchgeführt werden. 1. Vorbereitung des ergänzt adjuvante Wiegen Sie 30 mg Mycobacterium tuberculosis (Difco Katalog # 231141) und in einen Mörser. Machen Sie in dünne Pulver ohne Druck zu hart, um zu vermeiden brechen die Bakterien. 10 ml komplettem Freund-Adjuvans H37Ra (Difco Katalog # 231131) und mischen. Dies ergänzt adjuvante enthält nun 4 mg / ml Mycobacterium tuberculosis. Transfer in ein Rohr. 2. Vorbereitung der Rückenmark-Homogenat Sammeln Rückenmark von DA-Ratten (Alter / Geschlecht gleichgültig) und bei -80 ° C eingefroren Wiegen Sie die gefrorenen Rückenmark haben genug, um 1:1 (Gewicht: Volumen) mischen sich mit den ergänzten Adjuvans. Mince das Rückenmark so fein wie möglich mit einer Rasierklinge, um den Mörtel in Abschnitt 1 zu übertragen, und eine Paste mit dem Pistill. 3. Herstellung der Emulsion <ol><li> Legen Sie ergänzt komplettem Freund-Adjuvans in einem Rohr. Vortex mit hoher Geschwindigkeit. Fügen Sie die Rückenmark-Homogenat tropfenweise unter Vortexen. Wenn alle Rückenmark-Homogenat hinzugefügt wird, Wirbel für weitere 5 Minuten. Die Mischung sollte wiederum hellrosa. </li><li> Legen Sie die Emulsion in einem 5 ml Glasspritze und verknüpfen es mit weiteren 5 ml Glasspritze mit einer 18G-Brücke (Fisher Katalog # 14-825-17L). Senden Sie die Emulsion aus einer Spritze auf der anderen, bis sie hart (5-10 min) wird. Wenn mehr als 5 ml Emulsion hergestellt wird, nutzen mehrere 5 ml Spritzen KEINE größere Spritzen. </li><li> Die Emulsion kann in die Spritzen bei 4 ° C für 3 Wochen gelagert. Ich empfehle bereitet es mindestens 12 Stunden im Voraus zu überprüfen, ob sie nicht brechen. Es sollte auch weiterhin dick und nicht getrennt in 2 Phasen. Die Farbe wird über Nacht ändern, um eine hellgelbe oder beige. </li></ol> 4. Die Immunisierung der Ratten <ol><li> Mix der Emulsion in die Spritzen für ein paar Minuten und Transfer zum Spritzen für die Injektion verwendet. Inject 200 ul subkutan in die eigentliche Basis des Schwanzes mit 23G Nadeln und 3 ml Luer-Lock Spritzen unter kurzfristigen Anästhesie. </li><li> Die Empfänger sind 7-9 Wochen alte weibliche DA-Ratten. Wir bekommen unsere Ratten von Harlan-Sprague Dawley. </li><li> Ratten sollte zweimal täglich beobachtet und gewogen täglich. Klinische Symptome sind 7-15 Tage nach der Injektion der Emulsion zu erwarten. </li></ol> Klinische Wertung: 0: keine Krankheit 0,5: distalen schlaffen Schwanz 1: schlaffe Schwanz 2: mild Paraparese, Ataxie 3: mäßige Paraparese, die Ratten Reisen von Zeit zu Zeit 3,5: ein Hinterbein des Körpers ist gelähmt, die andere bewegt sich 4: komplette Hinterbein Lähmung 5: komplette Hinterbein Lähmung und Inkontinenz 6: moribund, Atembeschwerden, nicht essen oder trinken. Euthanize sofort.

CB und KGC sind Mitbegründer und Berater für Airmid Inc.

Es gibt ein paar wichtige Überlegungen für dieses Protokoll. Genetik der DA-Ratten wird etwas anders sein je nachdem, welche Züchter werden sie von gekauft. Diese Unterschiede können Zu-oder Abnahme der Empfindlichkeit von Ratten die Induktion EAE. Wenn Ihr Ratten anfälliger für EAE sind, werden Sie wollen, um die Stärke der Immunisierung unter Verwendung von nicht-ergänzten komplettem Freund-Adjuvans oder sogar unvollständig Freunds Adjuvans reduzieren. Sie können sich auch die Verringerung der Menge der Emulsion injizier...

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<td>Complete Freund's Adjuvant</td>
<td>Reagent</td>
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<td>Manufactured by Difco</td>
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<td>Mycobacterium tuberculosis H37Ra</td>
<td>Reagent</td>
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<td>DA rat</td>
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<td>Females, 7-9 weeks old.</td>
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<td>Glass syringes - 5 ml</td>
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<td>Luer Lok plastic syringes - 3 ml</td>
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<td>Needles - 20G 1 1/2</td>
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induction and clinical scoring of chronic-relapsing experimental autoimmune encephalomyelitis

Multiple Sklerose (MS) ist eine chronische entzündliche Erkrankung des zentralen Nervensystems (ZNS), die häufig bei jungen Erwachsenen. Es wird durch Demyelinisierung und Gliazellen zu erschrecken in Bereichen des Gehirns und des Rückenmarks verbreitet charakterisiert. Diese Läsionen verändern Nervenleitung und induzieren die zu Beeinträchtigungen führende neurologische Defizite, die mit der Lage der demyelinisierte Plaques im ZNS (zB Paraparese, Lähmung, Erblindung, Inkontinenz) variieren. Die experimentelle autoimmune Enzephalomyelitis (EAE) ist ein Modell für MS. EAE wurde zum ersten Mal versehentlich in den Menschen während der Tollwutimpfung induziert, mit Viren auf Kaninchen Rückenmark gewachsen. Rückstände von Rückenmark injiziert mit dem inaktivierten Virus induzierten ZNS Erkrankungen. Nach diesen Beobachtungen wurde ein erstes Modell der EAE in nicht-menschlichen Primaten mit einer ZNS-Homogenat durch Flüsse und Schwenther in 1935 immunisiert beschrieben. EAE ist seitdem in einer Vielzahl von Arten erzeugt worden und kann verschiedene Kurse je nach Art / Stamm und immunisierende Antigen verwendet folgen. Zum Beispiel Immunisierung Lewis Ratten mit Myelin basisches Protein in Emulsion mit Adjuvans induziert eine akute Modell der EAE, während das gleiche Antigen induziert eine chronische Krankheit bei Meerschweinchen. Die EAE-Modell beschrieben ist hier durch die Immunisierung von DA Ratten gegen DA Ratte Rückenmark in Emulsion in komplettem Freund-Adjuvans induziert. Ratten entwickeln eine aufsteigende schlaffe Lähmung innerhalb von 7-14 Tagen post-Immunisierung. Klinische Symptome folgen einem schubförmigen Verlauf über mehrere Wochen. Pathologie zeigt große Immunsystem Infiltrate im ZNS und Demyelinisierung Plaques. Besondere Überlegungen für die Betreuung von Tieren mit EAE sind am Ende des Videos beschrieben.

Watch this Scientific Journal Video about Induction and Clinical Scoring of Chronic-Relapsing Experimental Autoimmune Encephalomyelitis at JoVE.com

Induction and Clinical Scoring of Chronic-Relapsing Experimental Autoimmune Encephalomyelitis

Dieses Video zeigt die Induktion und klinischen Scoring von einem Tiermodell der Multiplen Sklerose: chronisch-rezidivierende experimentellen autoimmunen Enzephalomyelitis in DA-Ratten. Die Krankheit, durch die Immunisierung von Ratten mit einer Emulsion, die ganze Ratte Rückenmark und komplettem Freund-Adjuvans induziert, weist klinische Symptome ähnlich der menschlichen Erkrankung.

Induction and Clinical Scoring von chronisch-rezidivierende experimentellen autoimmunen Enzephalomyelitis

Multiple sclerosis (MS) is a chronic inflammatory disease of the central nervous system (CNS) that commonly affects young adults. It is characterized by ...

induction-clinical-scoring-chronic-relapsing-experimental-autoimmune

Research

JoVE Journal

Biology

17.9K Aufrufe.  University of California, Irvine (UCI). Dieses Video zeigt die Induktion und klinischen Scoring von einem Tiermodell der Multiplen Sklerose: chronisch-rezidivierende experimentellen autoimmunen Enzephalomyelitis in DA-Ratten. Die Krankheit, durch die Immunisierung von Ratten mit einer Emulsion, die ganze Ratte Rückenmark und komplettem Freund-Adjuvans induziert, weist klinische Symptome ähnlich der menschlichen Erkrankung.

Serie: Induction and Clinical Scoring of Chronic-Relapsing Experimental Autoimmune Encephalomyelitis

Experimental Approaches to Tissue Engineering

Experimentelle Ansätze zur Tissue Engineering

Forschung

Biologie

Induction and Monitoring of Adoptive Delayed-Type Hypersensitivity in Rats

Delayed type hypersensitivity (DTH) is an inflammatory reaction mediated by CCR7- effector memory T lymphocytes that infiltrate the site of injection of an antigen against which the immune system has been primed. The inflammatory reaction is characterized by redness and swelling of the site of antigenic challenge. It is a convenient model to determine the in vivo efficacy of immunosuppressants. Cutaneous DTH can be induced either by adoptive transfer of antigen-specific T lymphocytes or by active immunization with an antigen, and subsequent intradermal challenge with the antigen to induce the inflammatory reaction in a given skin area. DTH responses can be induced to various antigens, for example ovalbumin, tuberculin, tetanus toxoid, or keyhole limpet hemocyanin. Such reactions can also be induced against autoantigen, for example to myelin basic protein (MBP) in rats with experimental autoimmune encephalomyelitis induced with MBP, an animal model for multiple sclerosis (1).
Here we demonstrate how to induce an adoptive DTH reaction in Lewis rats. We will first stimulate ovalbumin-specific T cells in vitro and inject these activated cells intraperitoneally to naive rats. After allowing the cells to equilibrate in vivo for 2 days, we will challenge the rats with ovalbumin in the pinna of one ear, while the other ear wil receive saline. The inflammatory reaction will be visible 3-72 hours later and ear thickness will be measured as an indication of DTH severity.

Delayed type hypersensitivity (DTH) is an inflammatory reaction mediated by CCR7- effector memory T (TEM) lymphocytes. Here we demonstrate how to activate antigen-specific TEM cells, induce adoptive DTH in Lewis rats and monitor the inflammatory response.

Induktion und Überwachung von Adoptive Allergien vom verzögerten Typ in Ratten

Delayed type hypersensitivity (DTH) is an inflammatory reaction mediated by CCR7- effector memory T lymphocytes that infiltrate the site of injection of ...

The Ladder Rung Walking Task: A Scoring System and its Practical Application.

Progress in the development of animal models for/stroke, spinal cord injury, and other neurodegenerative disease requires tests of high sensitivity to elaborate distinct aspects of motor function and to determine even subtle loss of movement capacity. To enhance efficacy and resolution of testing, tests should permit qualitative and quantitative measures of motor function and be sensitive to changes in performance during recovery periods. The present study describes a new task to assess skilled walking in the rat to measure both forelimb and hindlimb function at the same time. Animals are required to walk along a horizontal ladder on which the spacing of the rungs is variable and is periodically changed. Changes in rung spacing prevent animals from learning the absolute and relative location of the rungs and so minimize the ability of the animals to compensate for impairments through learning. In addition, changing the spacing between the rungs allows the test to be used repeatedly in long-term studies. Methods are described for both quantitative and qualitative description of both fore- and hindlimb performance, including limb placing, stepping, co-ordination. Furthermore, use of compensatory strategies is indicated by missteps or compensatory steps in response to another limb&#x2019;s misplacement. 

The ladder rung walking task is a new test to assess skilled walking and measure both forelimb and hindlimb placing, stepping, and inter-limb co-ordination.

Die Leitersprosse Walking-Aufgabe: Ein Scoring-System und seine praktische Anwendung.

Progress in the development of animal models for/stroke, spinal cord injury, and other neurodegenerative disease requires tests of high sensitivity to ...

Live Cell Calcium Imaging Combined with siRNA Mediated Gene Silencing Identifies Ca2+ Leak Channels in the ER Membrane and their Regulatory Mechanisms

In mammalian cells, the endoplasmic reticulum (ER) plays a key role in protein biogenesis as well as in calcium signalling1. The heterotrimeric Sec61 complex in the ER membrane provides an aqueous path for newly-synthesized polypeptides into the lumen of the ER. Recent work from various laboratories suggested that this heterotrimeric complex may also form transient Ca2+ leak channels2-8. The key observation for this notion was that release of nascent polypeptides from the ribosome and Sec61 complex by puromycin leads to transient release of Ca2+ from the ER. Furthermore, it had been observed in vitro that the ER luminal protein BiP is involved in preventing ion permeability at the level of the Sec61 complex9,10. We have established an experimental system that allows us to directly address the role of the Sec61 complex as potential Ca2+ leak channel and to characterize its putative regulatory mechanisms11-13. This system combines siRNA mediated gene silencing and live cell Ca2+ imaging13. Cells are treated with siRNAs that are directed against the coding and untranslated region (UTR), respectively, of the SEC61A1 gene or a negative control siRNA. In complementation analysis, the cells are co-transfected with an IRES-GFP vector that allows the siRNA-resistant expression of the wildtype SEC61A1 gene. Then the cells are loaded with the ratiometric Ca2+-indicator FURA-2 to monitor simultaneously changes in the cytosolic Ca2+ concentration in a number of cells via a fluorescence microscope. The continuous measurement of cytosolic Ca2+ also allows the evaluation of the impact of various agents, such as puromycin, small molecule inhibitors, and thapsigargin on Ca2+ leakage. This experimental system gives us the unique opportunities to i) evaluate the contribution of different ER membrane proteins to passive Ca2+ efflux from the ER in various cell types, ii) characterize the proteins and mechanisms that limit this passive Ca2+ efflux, and iii) study the effects of disease linked mutations in the relevant components.

Live Cell Calcium Imaging Combined with siRNA Mediated Gene Silencing Identifies Ca2+ Leak Channels in the ER Membrane and their Regulatory Mechanisms

The endoplasmic reticulum plays a key role in protein biogenesis and in calcium homeostasis. We have established an experimental system that allows us to address the role of Ca2+ leak channels and to characterize their putative regulatory mechanisms. This system involves siRNA mediated gene silencing and live cell Ca2+ imaging.

Live Cell Imaging mit Calcium siRNA vermittelte Gene Silencing Combined Identifiziert Ca 2 + Leck-Kanäle in der ER-Membran und deren Regulationsmechanismen

In mammalian cells, the endoplasmic reticulum (ER) plays a key role in protein biogenesis as well as in calcium signalling1. The heterotrimeric Sec61 ...

Induction and Testing of Hypoxia in Cell Culture

Hypoxia is defined as the reduction or lack of oxygen in organs, tissues, or cells. This decrease of oxygen tension can be due to a reduced supply in oxygen (causes include insufficient blood vessel network, defective blood vessel, and anemia) or to an increased consumption of oxygen relative to the supply (caused by a sudden higher cell proliferation rate). Hypoxia can be physiologic or pathologic such as in solid cancers 1-3, rheumatoid arthritis, atherosclerosis etc&#x2026; Each tissues and cells have a different ability to adapt to this new condition. During hypoxia, hypoxia inducible factor alpha (HIF) is stabilized and regulates various genes such as those involved in angiogenesis or transport of oxygen 4. The stabilization of this protein is a hallmark of hypoxia, therefore detecting HIF is routinely used to screen for hypoxia 5-7. 
In this article, we propose two simple methods to induce hypoxia in mammalian cell cultures and simple tests to evaluate the hypoxic status of these cells.

Here we propose simple methods to induce hypoxia in cell cultures and simple tests to evaluate the hypoxic status of the cultures.

Induktion und Prüfung von Hypoxie in der Zellkultur

Hypoxia is defined as the reduction or lack of oxygen in organs, tissues, or cells.  This decrease of oxygen tension can be due to a reduced supply in ...

Ovariectomy and 17&beta;-estradiol Replacement in Rats and Mice: A Visual Demonstration

Estrogens are a family of female sexual hormones with an exceptionally wide spectrum of effects. When rats and mice are used in estrogen research they are commonly ovariectomized in order to ablate the rapidly cycling hormone production, replacing the 17&beta;-estradiol exogenously. There is, however, lack of consensus regarding how the hormone should be administered to obtain physiological serum concentrations. This is crucial since the 17&beta;-estradiol level/administration method profoundly influences the experimental results1-3. We have in a series of studies characterized the different modes of 17&beta;-estradiol administration, finding that subcutaneous silastic capsules and per-oral nut-cream Nutella are superior to commercially available slow-release pellets (produced by the company Innovative Research of America) and daily injections in terms of producing physiological serum concentrations of 17&beta;-estradiol4-6. Amongst the advantages of the nut-cream method, that previously has been used for buprenorphine administration7, is that when used for estrogen administration it resembles peroral hormone replacement therapy and is non-invasive. The subcutaneous silastic capsules are convenient and produce the most stable serum concentrations. This video article contains step-by-step demonstrations of ovariectomy and 17&beta;-estradiol hormone replacement by silastic capsules and peroral Nutella in rats and mice, followed by a discussion of important aspects of the administration procedures.

Ovariectomy and 17&amp;#946;-estradiol Replacement in Rats and Mice: A Visual Demonstration

Ovariectomy and subsequent replacement of 17&beta;-estradiol by means of silastic capsules and peroral Nutella are demonstrated in rats and mice.

Ovariektomie und 17β-Estradiol Ersatz bei Ratten und Mäusen: Die bildliche Darstellung

Estrogens are a family of female sexual hormones with an exceptionally wide spectrum of effects. When rats and mice are used in estrogen research they are ...

An Analytical Tool that Quantifies Cellular Morphology Changes from Three-dimensional Fluorescence Images

The most common software analysis tools available for measuring fluorescence images are for two-dimensional (2D) data that rely on manual settings for inclusion and exclusion of data points, and computer-aided pattern recognition to support the interpretation and findings of the analysis. It has become increasingly important to be able to measure fluorescence images constructed from three-dimensional (3D) datasets in order to be able to capture the complexity of cellular dynamics and understand the basis of cellular plasticity within biological systems. Sophisticated microscopy instruments have permitted the visualization of 3D fluorescence images through the acquisition of multispectral fluorescence images and powerful analytical software that reconstructs the images from confocal stacks that then provide a 3D representation of the collected 2D images. Advanced design-based stereology methods have progressed from the approximation and assumptions of the original model-based stereology1 even in complex tissue sections2. Despite these scientific advances in microscopy, a need remains for an automated analytic method that fully exploits the intrinsic 3D data to allow for the analysis and quantification of the complex changes in cell morphology, protein localization and receptor trafficking. 
Current techniques available to quantify fluorescence images include Meta-Morph (Molecular Devices, Sunnyvale, CA) and Image J (NIH) which provide manual analysis. Imaris (Andor Technology, Belfast, Northern Ireland) software provides the feature MeasurementPro, which allows the manual creation of measurement points that can be placed in a volume image or drawn on a series of 2D slices to create a 3D object. This method is useful for single-click point measurements to measure a line distance between two objects or to create a polygon that encloses a region of interest, but it is difficult to apply to complex cellular network structures. Filament Tracer (Andor) allows automatic detection of the 3D neuronal filament-like however, this module has been developed to measure defined structures such as neurons, which are comprised of dendrites, axons and spines (tree-like structure). This module has been ingeniously utilized to make morphological measurements to non-neuronal cells3, however, the output data provide information of an extended cellular network by using a software that depends on a defined cell shape rather than being an amorphous-shaped cellular model. To overcome the issue of analyzing amorphous-shaped cells and making the software more suitable to a biological application, Imaris developed Imaris Cell. This was a scientific project with the Eidgen&ouml;ssische Technische Hochschule, which has been developed to calculate the relationship between cells and organelles. While the software enables the detection of biological constraints, by forcing one nucleus per cell and using cell membranes to segment cells, it cannot be utilized to analyze fluorescence data that are not continuous because ideally it builds cell surface without void spaces. To our knowledge, at present no user-modifiable automated approach that provides morphometric information from 3D fluorescence images has been developed that achieves cellular spatial information of an undefined shape (Figure 1). 
We have developed an analytical platform using the Imaris core software module and Imaris XT interfaced to MATLAB (Mat Works, Inc.). These tools allow the 3D measurement of cells without a pre-defined shape and with inconsistent fluorescence network components. Furthermore, this method will allow researchers who have extended expertise in biological systems, but not familiarity to computer applications, to perform quantification of morphological changes in cell dynamics.

We developed a software platform that utilizes Imaris Neuroscience, ImarisXT and MATLAB to measure the changes in morphology of an undefined shape taken from three-dimensional confocal fluorescence of single cells. This novel approach can be used to quantify changes in cell shape following receptor activation and therefore represents a possible additional tool for drug discovery.

Ein analytisches Werkzeug, das zelluläre Morphologie Änderungen von Dreidimensionale Fluorescence Images Quantifiziert

The most common software analysis tools available for measuring fluorescence images are for two-dimensional (2D) data that rely on manual settings for ...

Isolation and Kv Channel Recordings in Murine Atrial and Ventricular Cardiomyocytes

KCNE genes encode for a small family of Kv channel ancillary subunits that form heteromeric complexes with Kv channel alpha subunits to modify their functional properties. Mutations in KCNE genes have been found in patients with cardiac arrhythmias such as the long QT syndrome and/or atrial fibrillation. However, the precise molecular pathophysiology that leads to these diseases remains elusive. In previous studies the electrophysiological properties of the disease causing mutations in these genes have mostly been studied in heterologous expression systems and we cannot be sure if the reported effects can directly be translated into native cardiomyocytes. In our laboratory we therefore use a different approach. We directly study the effects of KCNE gene deletion in isolated cardiomyocytes from knockout mice by cellular electrophysiology - a unique technique that we describe in this issue of the Journal of Visualized Experiments. The hearts from genetically engineered KCNE mice are rapidly excised and mounted onto a Langendorff apparatus by aortic cannulation. Free Ca2+ in the myocardium is bound by EGTA, and dissociation of cardiac myocytes is then achieved by retrograde perfusion of the coronary arteries with a specialized low Ca2+ buffer containing collagenase. Atria, free right ventricular wall and the left ventricle can then be separated by microsurgical techniques. Calcium is then slowly added back to isolated cardiomyocytes in a multiple step comprising washing procedure. Atrial and ventricular cardiomyocytes of healthy appearance with no spontaneous contractions are then immediately subjected to electrophysiological analyses by patch clamp technique or other biochemical analyses within the first 6 hours following isolation.

Kv channel dysfunction is associated with cardiac arrhythmias. In order to study the molecular mechanisms that lead to such arrhythmias we utilize a systematic protocol for isolation of atrial and ventricular cardiomyocytes from Kv channel ancillary subunit knockout mice. Isolated cardiomyocytes can then immediately be used for cellular electrophysiological studies, biochemical or immunofluorescence (IF) assays.

Isolierung und Kv Kanal Recordings in Murine atriale und ventrikuläre Kardiomyozyten

KCNE genes encode for a small family of Kv channel ancillary subunits that form heteromeric complexes with Kv channel alpha subunits to modify their ...

Induction and Analysis of Epithelial to Mesenchymal Transition

Epithelial to mesenchymal transition (EMT) is essential for proper morphogenesis during development. Misregulation of this process has been implicated as a key event in fibrosis and the progression of carcinomas to a metastatic state. Understanding the processes that underlie EMT is imperative for the early diagnosis and clinical control of these disease states. Reliable induction of EMT in vitro is a useful tool for drug discovery as well as to identify common gene expression signatures for diagnostic purposes. Here we demonstrate a straightforward method for the induction of EMT in a variety of cell types. Methods for the analysis of cells pre- and post-EMT induction by immunocytochemistry are also included. Additionally, we demonstrate the effectiveness of this method through antibody-based array analysis and migration/invasion assays.

A straightforward method is described for the induction of epithelial to mesenchymal transition (EMT) in a variety of cell types. Methods for the analysis of cells in EMT states by immunocytochemistry are included.

Induktion und Analyse von epithelialen zu mesenchymalen Transition

Epithelial  to mesenchymal transition (EMT) is essential for proper morphogenesis during  development. Misregulation of this  process has been implicated ...

Screening for Functional Non-coding Genetic Variants Using Electrophoretic Mobility Shift Assay (EMSA) and DNA-affinity Precipitation Assay (DAPA)

Population and family-based genetic studies typically result in the identification of genetic variants that are statistically associated with a clinical disease or phenotype. For many diseases and traits, most variants are non-coding, and are thus likely to act by impacting subtle, comparatively hard to predict mechanisms controlling gene expression. Here, we describe a general strategic approach to prioritize non-coding variants, and screen them for their function. This approach involves computational prioritization using functional genomic databases followed by experimental analysis of differential binding of transcription factors (TFs) to risk and non-risk alleles. For both electrophoretic mobility shift assay (EMSA) and DNA affinity precipitation assay (DAPA) analysis of genetic variants, a synthetic DNA oligonucleotide (oligo) is used to identify factors in the nuclear lysate of disease or phenotype-relevant cells. For EMSA, the oligonucleotides with or without bound nuclear factors (often TFs) are analyzed by non-denaturing electrophoresis on a tris-borate-EDTA (TBE) polyacrylamide gel. For DAPA, the oligonucleotides are bound to a magnetic column and the nuclear factors that specifically bind the DNA sequence are eluted and analyzed through mass spectrometry or with a reducing sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) followed by Western blot analysis. This general approach can be widely used to study the function of non-coding genetic variants associated with any disease, trait, or phenotype.

Screening for Functional Non-coding Genetic Variants Using Electrophoretic Mobility Shift Assay EMSA and DNA-affinity Precipitation Assay DAPA

We present a strategic plan and protocol for identifying non-coding genetic variants affecting transcription factor (TF) DNA binding. A detailed experimental protocol is provided for electrophoretic mobility shift assay (EMSA) and DNA affinity precipitation assay (DAPA) analysis of genotype-dependent TF DNA binding.

Screening für Funktionelle nichtkodierenden genetischen Varianten Mit elektrophoretische Mobilitäts-Shift-Assay (EMSA) und DNA-Affinitätspräzipitation Assay (DAPA)

Population and family-based genetic studies typically result in the identification of genetic variants that are statistically associated with a clinical ...