Авторы благодарят доктора Энн Гиббон, доктор Кристин Mackay и всех сотрудников Монаш Услуги животных и физиологии животных дома. Авторы выражают благодарность Джереми Вайнштейн и команда Mcube для видео и анимации production.a

<ol><li> Овец постился в течение 24 часов, но без ограничений разрешено воды объявлений </li><li> Овец взвешивают. </li><li> Anaestheisa индуцирована внутривенного введения thiopentone (20mg/kg). </li><li> Профилактическое внутривенное введение антибиотиков может быть дано. </li><li> Интубация трахеи выполняется с помощью размера от 7 до 9 манжетой эндотрахеальной трубки. </li><li> Анестезия является maintainted с изофлуран, 1-3%, в 100% кислорода при расходе 2 л в минуту. </li><li> Наблюдения производятся и уровень анестезии контролируется постоянно. </li><li> Хирургические инструменты, необходимые для этой операции включают в себя: зубчатые щипцы, без зубчатых щипцов, Metzenbaum ножниц, Майо ножницы, иглодержатели, скальпеля ручку (длинные и короткие), Керрисон rongeurs, гипофиза rongeurs, пневматические хирургические дрели, сверла и охранников, нервные крючки (тупой и микро крюк), лифт (то есть, Уотсон Чейни), изолированный моно полярных и биполярных электрохирургических щипцы, позвоночный отвлекающих тела (т.е. 16 мм контактов Каспар), сверлить отвлечение контактный и сверлить рукав, 2-0, 3-0 и угловые кюреты , размер 7 и 10 французских присоски и всасывающая трубка, Langenback преднатяжителями и самоудерживающий шейки преднатяжителями. </li><li> Области шеи является побрился и промывают раствором моющего средства бетадин. </li><li> Животное помещается в положении лежа на операционном столе и шеи мягко продлен. </li><li> Кожи отмечается с помощью иглы и рентгеновского берется подтвердить C3 / 4. </li><li> Шея затем нацелен использованием алкогольных хлоргексидин, алкогольные йода и 70% этанола в воде. </li><li> Окончатый квадратных драпировка используется для хирургической сайта и большая площадь драпировка для накладных таблице. </li><li> Операционного микроскопа или лупы увеличение и лампы, которые используются для хирургической операции. </li><li> Надрез скальпелем на месте ранее выявленных использованием рентгеновского </li><li> Далее, рассечение через инвестирование слоев осуществляется с помощью ножниц и самолет разработан медиальнее сонной оболочки и сбоку от трахеи и пищевода. </li><li> Гемостаз поддерживается посредством процедуры с использованием диатермии </li><li> После позвоночника находится, по средней линии должны быть пальпируется и уход должны быть приняты для обеспечения того, чтобы это опровержение медиальнее сонной артерии и сбоку от трахеи и пищевода. </li><li> Штыками иглы затем вставляется диск (это предотвращает случайное продвижение которые могли бы риск повреждения спинного мозга). Квадратных драпировка используется для покрытия хирургических сайта. Перед интраоперационного рентген берется подтвердить C3 / 4 диска уровне. </li><li> Контакты Каспер отвлечение Затем вставляются в С3 и С4 соответственно. </li><li> Диск надрезанные с 15-лезвие скальпеля. </li><li> Отвлекающих затем вставляется и дискового пространства, мягко отвлекаться друг от друга. </li><li> Дискэктомии затем выполняется с помощью rongeurs и Керрисон rongeurs. Сверла и кюреты также может оказать помощь. </li><li> Концевые затем подготовлен с использованием буровых и кюреты пока кровотечение кости визуализируется. </li><li> Задней продольной связки могут быть открыты в данный момент, в зависимости от эксперимента. Тупым крючком и 1мм Керрисон лучше всего использовать для этой процедуры. </li><li> Рана то орошаемых с физиологическим раствором, до введения клетке межтелового или тестового материала. </li><li> Отвлечение расслаблен объявление устройства проверены, чтобы убедиться, что он надежно закреплен. </li><li> Отвлечение штифты удаляют воск и кости могут быть вставлены для достижения гемостаза в кости отверстия. </li><li> Мышцы Лонга кишечной закрыта с Викрил. Многоуровневая закрытия Затем выполняется, желательно с подкожный шов, чтобы избежать необходимости удаления шва. </li><li> Xylocaine проникли вокруг места операции </li><li> Газа anaestheitc выключен, и, когда происходит спонтанное дыхание и глотание признаки очевидны, эндотрахеальная трубка будет удалена. </li><li> Животное затем позволили восстановить в метаболической клетке под постоянным наблюдением. </li><li> Важно, что животное находится в вертикальном положении ноги скрываются под ней, чтобы предотвратить срыгивание. </li><li> Как только животное стоит (обычно в течение 1 часа) пищи и воды, может быть вновь. </li><li> Закрыть мониторинг осуществляется в течение 24 часов и продолжалось в течение 1 недели. </li><li> Обезболивание ведется с Фентанил патч для 3-х дней после операции. </li></ol>

<ol>
	<li>Angevine, P. D., Arons, R. R., McCormick, 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=National+and+regional+rates+and+variation+of+cervical+discectomy+with+and+without+anterior+fusion,+1990-1999.">National and regional rates and variation of cervical discectomy with and without anterior fusion, 1990-1999.</a> Spine. 28, 931-940 (2003).</li><li>Cloward, R. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+anterior+approach+for+removal+of+ruptured+cervical+disks.">The anterior approach for removal of ruptured cervical disks.</a> J Neurosurg. 15, 602-617 (1958).</li><li>Jacobs, W. C., Anderson, P. G., Limbeek, J., Willems, P. C., Pavlov, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single+or+double-level+anterior+interbody+fusion+techniques+for+cervical+degenerative+disc+disease.">Single or double-level anterior interbody fusion techniques for cervical degenerative disc disease.</a> Cochrane Database Syst Rev. , CD004958-CD004958 (2004).</li><li>Kandziora, F., Pflugmacher, R., Scholz, M., Schnake, K., Lucke, M., Schr&#246;der, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+between+sheep+and+human+cervical+spines:+an+anatomic,+radiographic,+bone+mineral+density,+and+biomechanical+study.">Comparison between sheep and human cervical spines: an anatomic, radiographic, bone mineral density, and biomechanical study.</a> Spine. 26, 1028-1037 (2001).</li><li>Patil, P. G., Turner, D. A., 	Pietrobon, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=National+trends+in+surgical+procedures+for+degenerative+cervical+spine+disease:+1990-2000.">National trends in surgical procedures for degenerative cervical spine disease: 1990-2000.</a> Neurosurgery. 57, 753-758 (2005).</li><li>Smith, G. W., Robinson, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+treatment+of+certain+cervical-spine+disorders+by+anterior+removal+of+the+intervertebral+disc+and+interbody+fusion.">The treatment of certain cervical-spine disorders by anterior removal of the intervertebral disc and interbody fusion.</a> J Bone Joint Surg Am. 40-A, 607-624 (1958).</li></ol>

Есть несколько источников изменчивости, чтобы рассмотреть при планировании и проведении передней шейной дискэктомии и синтеза. Таким образом, для последовательности модели, животные должны быть того же возраста и породы. Мы предпочитаем два года старый, гибрид Лестер Мерино овец. Есл?...

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<td>Marcain</td>
<td>&nbsp;</td>
<td>AstraZeneca</td>
<td>&nbsp;</td>
<td>0/5% Bupivicaine hydrochloride solution for injection 100mg in 20 mls</td>
</tr>
<tr>
<td>Thiopentone</td>
<td>&nbsp;</td>
<td>Illium</td>
<td>&nbsp;</td>
<td>Thiopentone sodium (sterile powder for injection) 5g</td>
</tr>
<tr>
<td>Issoranne</td>
<td>&nbsp;</td>
<td>Baxter</td>
<td>&nbsp;</td>
<td>Isoflurane BP 100%</td>
</tr>
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<td>Chlorehxidine</td>
<td>&nbsp;</td>
<td>Jurox</td>
<td>&nbsp;</td>
<td>40mg/ml Chlorhexidine glucoronate</td>
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<td>PVD Iodine Solution</td>
<td>&nbsp;</td>
<td>Apex</td>
<td>&nbsp;</td>
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anterior cervical discectomy and fusion in the ovine model

Передняя дискэктомии шейного и синтеза (ACDF) является наиболее распространенной хирургической операции шейки радикулопатии и / или миелопатии у больных, которые не смогли консервативное лечение<sup&gt; 1,5</sup&gt;. Так как операция была впервые описана Cloward<sup&gt; 2</sup&gt; И Смит и Робинсон<sup&gt; 6</sup&gt; В 1958 году, различные усовершенствования в технике, взяточничество материалов и имплантатов были сделаны<sup&gt; 3</sup&gt;. В частности, существует потребность в безопасной остеоиндуктивных агентов, которые могли бы воспользоваться отдельных пациентов. Овец модели было показано, что анатомические, биомеханические, плотность костной ткани и радиологических свойств, которые похожи на человеческие коллегой, наиболее близким уровнем будучи C3 / 4<sup&gt; 4</sup&gt;. Поэтому идеальная модель, в которой доклинические исследования могут быть выполнены. В частности, эта методика может быть полезной для исследователей, заинтересованных в оценке различных устройств и биопрепаратов, в том числе стволовых клеток с целью возможного применения в человеческой хирургии позвоночника.

Watch this Scientific Journal Video about Anterior Cervical Discectomy and Fusion in the Ovine Model at JoVE.com

Anterior Cervical Discectomy and Fusion in the Ovine Model

Это видео демонстрирует технику передней шейной дискэктомии и синтеза в бараньих модели.

Передняя шейки Дискэктомия и термоядерных в баранине модели

Anterior cervical discectomy and fusion (ACDF) is the most common surgical operation for cervical radiculopathy and/or myelopathy in patients who have ...

anterior-cervical-discectomy-and-fusion-in-the-ovine-model

Research

JoVE Journal

Biology

12.8K Views.  Monash University. Это видео демонстрирует технику передней шейной дискэктомии и синтеза в бараньих модели.

Video: Anterior Cervical Discectomy and Fusion in the Ovine Model

Analysis of SNARE-mediated Membrane Fusion Using an Enzymatic Cell Fusion Assay

The interactions of SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins on vesicles (v-SNAREs) and on target membranes (t-SNAREs) catalyze intracellular vesicle fusion1-4. Reconstitution assays are essential for dissecting the mechanism and regulation of SNARE-mediated membrane fusion5. In a cell fusion assay6,7, SNARE proteins are expressed ectopically at the cell surface. These "flipped" SNARE proteins drive cell-cell fusion, demonstrating that SNAREs are sufficient to fuse cellular membranes. Because the cell fusion assay is based on microscopic analysis, it is less efficient when used to analyze multiple v- and t-SNARE interactions quantitatively.
Here we describe a new assay8 that quantifies SNARE-mediated cell fusion events by activated expression of &beta;-galactosidase. Two components of the Tet-Off gene expression system9 are used as a readout system: the tetracycline-controlled transactivator (tTA) and a reporter plasmid that encodes the LacZ gene under control of the tetracycline-response element (TRE-LacZ). We transfect tTA into COS-7 cells that express flipped v-SNARE proteins at the cell surface (v-cells) and transfect TRE-LacZ into COS-7 cells that express flipped t-SNARE proteins at the cell surface (t-cells). SNARE-dependent fusion of the v- and t-cells results in the binding of tTA to TRE, the transcriptional activation of LacZ and expression of &beta;-galactosidase. The activity of &beta;-galactosidase is quantified using a colorimetric method by absorbance at 420 nm.
The vesicle-associated membrane proteins (VAMPs) are v-SNAREs that reside in various post-Golgi vesicular compartments10-15. By expressing VAMPs 1, 3, 4, 5, 7 and 8 at the same level, we compare their membrane fusion activities using the enzymatic cell fusion assay. Based on spectrometric measurement, this assay offers a quantitative approach for analyzing SNARE-mediated membrane fusion and for high-throughput studies.

We have developed a cell fusion assay that quantifies SNARE-mediated membrane fusion events by activated expression of &beta;-galactosidase.

Анализ SNARE-опосредованного слияния мембран помощью ферментативного анализа слияния клеток

The interactions of SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor)  proteins on vesicles (v-SNAREs) and on target membranes ...

Evaluation of Muscle Function of the Extensor Digitorum Longus Muscle Ex vivo and Tibialis Anterior Muscle In situ in Mice

Body movements are mainly provided by mechanical function of skeletal muscle. Skeletal muscle is composed of numerous bundles of myofibers that are sheathed by intramuscular connective tissues. Each myofiber contains many myofibrils that run longitudinally along the length of the myofiber. Myofibrils are the contractile apparatus of muscle and they are composed of repeated contractile units known as sarcomeres. A sarcomere unit contains actin and myosin filaments that are spaced by the Z discs and titin protein. Mechanical function of skeletal muscle is defined by the contractile and passive properties of muscle. The contractile properties are used to characterize the amount of force generated during muscle contraction, time of force generation and time of muscle relaxation. Any factor that affects muscle contraction (such as interaction between actin and myosin filaments, homeostasis of calcium, ATP/ADP ratio, etc.) influences the contractile properties. The passive properties refer to the elastic and viscous properties (stiffness and viscosity) of the muscle in the absence of contraction. These properties are determined by the extracellular and the intracellular structural components (such as titin) and connective tissues (mainly collagen) 1-2. The contractile and passive properties are two inseparable aspects of muscle function. For example, elbow flexion is accomplished by contraction of muscles in the anterior compartment of the upper arm and passive stretch of muscles in the posterior compartment of the upper arm. To truly understand muscle function, both contractile and passive properties should be studied.
The contractile and/or passive mechanical properties of muscle are often compromised in muscle diseases. A good example is Duchenne muscular dystrophy (DMD), a severe muscle wasting disease caused by dystrophin deficiency 3. Dystrophin is a cytoskeletal protein that stabilizes the muscle cell membrane (sarcolemma) during muscle contraction 4. In the absence of dystrophin, the sarcolemma is damaged by the shearing force generated during force transmission. This membrane tearing initiates a chain reaction which leads to muscle cell death and loss of contractile machinery. As a consequence, muscle force is reduced and dead myofibers are replaced by fibrotic tissues 5. This later change increases muscle stiffness 6. Accurate measurement of these changes provides important guide to evaluate disease progression and to determine therapeutic efficacy of novel gene/cell/pharmacological interventions. Here, we present two methods to evaluate both contractile and passive mechanical properties of the extensor digitorum longus (EDL) muscle and the contractile properties of the tibialis anterior (TA) muscle.

Evaluation of Muscle Function of the Extensor Digitorum Longus Muscle Ex vivo and Tibialis Anterior Muscle In situ in Mice

Changes in limb muscle contractile and passive mechanical properties are important biomarkers for muscle diseases. This manuscript describes physiological assays to measure these properties in the murine extensor digitorum longus and tibialis anterior muscles.

Оценка функции мышц-разгибателей мышц пальцев Longus<em&gt; EX VIVO</em&gt; И передней большеберцовой мышцы<em&gt; В месте</em&gt; У мышей

Body  movements are mainly provided by mechanical function of skeletal muscle. Skeletal  muscle is composed of numerous bundles of myofibers that are ...

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

The folding and assembly of proteins is essential for protein function, the long-term health of the cell, and longevity of the organism. Historically, the function and regulation of protein folding was studied in vitro, in isolated tissue culture cells and in unicellular organisms. Recent studies have uncovered links between protein homeostasis (proteostasis), metabolism, development, aging, and temperature-sensing. These findings have led to the development of new tools for monitoring protein folding in the model metazoan organism Caenorhabditis elegans. In our laboratory, we combine behavioral assays, imaging and biochemical approaches using temperature-sensitive or naturally occurring metastable proteins as sensors of the folding environment to monitor protein misfolding. Behavioral assays that are associated with the misfolding of a specific protein provide a simple and powerful readout for protein folding, allowing for the fast screening of genes and conditions that modulate folding. Likewise, such misfolding can be associated with protein mislocalization in the cell. Monitoring protein localization can, therefore, highlight changes in cellular folding capacity occurring in different tissues, at various stages of development and in the face of changing conditions. Finally, using biochemical tools ex vivo, we can directly monitor protein stability and conformation. Thus, by combining behavioral assays, imaging and biochemical techniques, we are able to monitor protein misfolding at the resolution of the organism, the cell, and the protein, respectively.

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

To study the relationship between protein homeostasis, stress and aging, we monitored changes in protein folding by following protein dysfunction, protein localization in the cell and protein stability at the organismal, cellular and protein levels, using the genetically tractable metazoan Caenorhabditis elegans as a model system.

The folding and assembly of proteins is essential for protein function, the long-term health of the cell, and longevity of the organism. Historically, the ...

The Assembly and Application of 'Shear Rings': A Novel Endothelial Model for Orbital, Unidirectional and Periodic Fluid Flow and Shear Stress

Deviations from normal levels and patterns of vascular fluid shear play important roles in vascular physiology and pathophysiology by inducing adaptive as well as pathological changes in endothelial phenotype and gene expression. In particular, maladaptive effects of periodic, unidirectional flow induced shear stress can trigger a variety of effects on several vascular cell types, particularly endothelial cells. While by now endothelial cells from diverse anatomic origins have been cultured, in-depth analyses of their responses to fluid shear have been hampered by the relative complexity of shear models (e.g., parallel plate flow chamber, cone and plate flow model). While these all represent excellent approaches, such models are technically complicated and suffer from drawbacks including relatively lengthy and complex setup time, low surface areas, requirements for pumps and pressurization often requiring sealants and gaskets, creating challenges to both maintenance of sterility and an inability to run multiple experiments. However, if higher throughput models of flow and shear were available, greater progress on vascular endothelial shear responses, particularly periodic shear research at the molecular level, might be more rapidly advanced. Here, we describe the construction and use of shear rings: a novel, simple-to-assemble, and inexpensive tissue culture model with a relatively large surface area that easily allows for a high number of experimental replicates in unidirectional, periodic shear stress studies on endothelial cells.

The Assembly and Application of &#039;Shear Rings&#039;: A Novel Endothelial Model for Orbital, Unidirectional and Periodic Fluid Flow and Shear Stress

Different levels and patterns of fluid shear are known to modulate endothelial gene expression, phenotype and susceptibility to disease. We discuss the assembly and use of 'shear rings': a model that produces unidirectional, periodic shear stress patterns. Shear rings are simple to assemble, economical and can produce high cell yields.

Ассамблея и применение &quot;Shear колец&quot;: Роман эндотелиальной Модель для Orbital, однонаправленные и периодического потока жидкости и напряжения сдвига

Deviations from normal levels and patterns of vascular fluid shear play important roles in vascular physiology and pathophysiology by inducing adaptive as ...

Expression of Fluorescent Fusion Proteins in Murine Bone Marrow-derived Dendritic Cells and Macrophages

Dendritic cells and macrophages are crucial cells that form the first line of defense against pathogens. They also play important roles in the initiation of an adaptive immune response. Experimental work with these cells is rather challenging. Their abundance in organs and tissues is relatively low. As a result, they cannot be isolated in large numbers. They are also difficult to transfect with cDNA constructs. In the murine model, these problems can be partially overcome by in vitro differentiation from bone marrow progenitors in the presence of M-CSF for macrophages or GM-CSF for dendritic cells. In this way, it is possible to obtain large amounts of these cells from very few animals. Moreover, bone marrow progenitors can be transduced with retroviral vectors carrying cDNA constructs during early stages of cultivation prior to their differentiation into bone marrow derived dendritic cells and macrophages. Thus, retroviral transduction followed by differentiation in vitro can be used to express various cDNA constructs in these cells. The ability to express ectopic proteins substantially extends the range of experiments that can be performed on these cells, including live cell imaging of fluorescent proteins, tandem purifications for interactome analyses, structure-function analyses, monitoring of cellular functions with biosensors and many others. In this article, we describe a detailed protocol for retroviral transduction of murine bone marrow derived dendritic cells and macrophages with vectors coding for fluorescently-tagged proteins. On the example of two adaptor proteins, OPAL1 and PSTPIP2, we demonstrate its practical application in flow cytometry and microscopy. We also discuss the advantages and limitations of this approach.

In this article, we provide a detailed protocol for the expression of fluorescent fusion proteins in murine bone marrow derived dendritic cells and macrophages. The method is based on the transduction of bone marrow progenitors with retroviral constructs followed by differentiation into macrophages and dendritic cells in vitro.

Выражение флуоресцентные синтез белков в мышиных костного мозга-производные дендритные клетки и макрофаги

Dendritic cells and macrophages are crucial cells that form the first line of defense against pathogens. They also play important roles in the initiation ...

Real Time In Vivo Tracking of Thymocytes in the Anterior Chamber of the Eye by Laser Scanning Microscopy

The purpose of the method being presented is to show, for the first time, the transplant of newborn thymi into the anterior eye chamber of isogenic adult mice for in vivo longitudinal real-time monitoring of thymocytes&#180; dynamics within a vascularized thymus segment. Following the transplantation, laser scanning microscopy (LSM) through the cornea allows in vivo noninvasive repeated imaging at cellular resolution level. Importantly, the approach adds to previous intravital T-cell maturation imaging models the possibility for continuous progenitor cell recruitment and mature T-cell egress recordings in the same animal. Additional advantages of the system are the transparency of the grafted area, permitting macroscopic rapid monitoring of the implanted tissue, and the accessibility to the implant allowing for localized in addition to systemic treatments. The main limitation being the volume of the tissue that fits in the reduced space of the eye chamber which demands for lobe trimming. Organ integrity is maximized by dissecting thymus lobes in patterns previously shown to be functional for mature T-cell production. The technique is potentially suited to interrogate a milieu of medically relevant questions related to thymus function that include autoimmunity, immunodeficiency and central tolerance; processes which remain mechanistically poorly defined. The fine dissection of mechanisms guiding thymocyte migration, differentiation and selection should lead to novel therapeutic strategies targeting developing T cells.

Real Time In Vivo Tracking of Thymocytes in the Anterior Chamber of the Eye by Laser Scanning Microscopy

The goal of the protocol is to show longitudinal intravital real-time tracking of thymocytes by laser scanning microscopy in thymic implants in the anterior chamber of the mouse eye. The transparency of the cornea and vascularization of the graft allows for continuously recording progenitor cell recruitment and mature T-cell egress.

В режиме реального времени в Vivo отслеживания тимоцитов в передней камере глаза лазерная сканирующая микроскопия

The purpose of the method being presented is to show, for the first time, the transplant of newborn thymi into the anterior eye chamber of isogenic adult ...

Effect of Fluorescent Proteins on Fusion Partners Using Polyglutamine Toxicity Assays in Yeast

For the investigation of protein localization and trafficking using live cell imaging, researchers often rely on fusing their protein of interest to a fluorescent reporter. The constantly evolving list of genetically encoded fluorescent proteins (FPs) presents users with several alternatives when it comes to fluorescent fusion design. Each FP has specific optical and biophysical properties that can affect the biochemical, cellular, and functional properties of the resulting fluorescent fusions. For instance, several FPs tend to form nonspecific oligomers that are susceptible to impede on the function of the fusion partner. Unfortunately, only a few methods exist to test the impact of FPs on the behavior of the fluorescent reporter. Here, we describe a simple method that enables the rapid assessment of the impact of FPs using polyglutamine (polyQ) toxicity assays in the budding yeast Saccharomyces cerevisiae. PolyQ-expanded huntingtin proteins are associated with the onset of Huntington&#39;s disease (HD), where the expanded huntingtin aggregates into toxic oligomers and inclusion bodies. The aggregation and toxicity of polyQ expansions in yeast are highly dependent on the sequences flanking the polyQ region, including the presence of fluorescent tags, thus providing an ideal experimental platform to study the impact of FPs on the behavior of their fusion partner.

This article describes protocols to assess the effect of fluorescent proteins on the aggregation and toxicity of misfolded polyglutamine expansion for the rapid evaluation of a newly uncharacterized fluorescent protein in the context of fluorescent reporters.

Влияние на Fusion партнеров с помощью анализов токсичности Хантингтона в дрожжи флуоресцентных белков

For the investigation of protein localization and trafficking using live cell imaging, researchers often rely on fusing their protein of interest to a ...

Mass Spectrometry Analysis to Identify Ubiquitylation of EYFP-tagged CENP-A (EYFP-CENP-A)

Studying the structure and the dynamics of kinetochores and centromeres is important in understanding chromosomal instability (CIN) and cancer progression. How the chromosomal location and function of a centromere (i.e., centromere identity) are determined and participate in accurate chromosome segregation is a fundamental question. CENP-A is proposed to be the non-DNA indicator (epigenetic mark) of centromere identity, and CENP-A ubiquitylation is required for CENP-A deposition at the centromere, inherited through dimerization between cell division, and indispensable to cell viability.
Here we describe mass spectrometry analysis to identify ubiquitylation of EYFP-CENP-A K124R mutant suggesting that ubiquitylation at a different lysine is induced because of the EYFP tagging in the CENP-A K124R mutant protein. Lysine 306 (K306) ubiquitylation in EYFP-CENP-A K124R was successfully identified, which corresponds to lysine 56 (K56) in CENP-A through mass spectrometry analysis. A caveat is discussed in the use of GFP/EYFP or the tagging of high molecular weight protein as a tool to analyze the function of a protein. Current technical limit is also discussed for the detection of ubiquitylated bands, identification of site-specific ubiquitylation(s), and visualization of ubiquitylation in living cells or a specific single cell during the whole cell cycle.
The method of mass spectrometry analysis presented here can be applied to human CENP-A protein with different tags and other centromere-kinetochore proteins. These combinatory methods consisting of several assays/analyses could be recommended for researchers who are interested in identifying functional roles of ubiquitylation.

Mass Spectrometry Analysis to Identify Ubiquitylation of EYFP-tagged CENP-A EYFP-CENP-A

CENP-A ubiquitylation is an important requirement for CENP-A deposition at the centromere, inherited through dimerization between cell division, and indispensable to cell viability. Here we describe mass spectrometry analysis to identify ubiquitylation of EYFP-tagged CENP-A (EYFP-CENP-A) protein.

Анализ масс-спектрометрии для выявления повсеместного ания метеоуожего тега CENP-A (EYFP-CENP-A)

Studying the structure and the dynamics of kinetochores and centromeres is important in understanding chromosomal instability (CIN) and cancer ...

Production of IgG Fusion Proteins Transiently Expressed in Nicotiana benthamiana

High demand for antibodies as therapeutic interventions for various infectious, metabolic, autoimmune, neoplastic, and other diseases creates a growing need in developing efficient methods for recombinant antibody production. As of 2019, there were more than 70 FDA-approved monoclonal antibodies, and there is exponential growth potential. Despite their promise, limiting factors for widespread use are manufacturing costs and complexity. Potentially, plants offer low-cost, safe, and easily scalable protein manufacturing&#160;strategies. Plants like Nicotiana benthamiana not only can correctly fold and assemble complex mammalian proteins but also can add critical post-translational modifications similar to those offered by mammalian cell cultures. In this work, by using native GFP and an acid-stable variant of green fluorescent protein (GFP) fused to human monoclonal antibodies, we were able to visualize the entire transient antibody expression and purification process from N. benthamiana plants. Depending on the experiment&#39;s purpose, native GFP fusion can ensure easier visualization during the expression phase in the plants, while acid-stable GFP fusion allows for visualization during downstream processing. This scalable and straightforward procedure can be performed by a single researcher to produce milligram quantities of highly pure antibody or antibody fusion proteins in a matter of days using only a few small plants. Such a technique can be extended to the visualization of any type of antibody purification process and potentially many other proteins, both in plant and other expression systems. Moreover, these techniques can benefit virtual instructions and be executed in a teaching laboratory by undergraduate students possessing minimal prior experience with molecular biology techniques, providing a foundation for project-based exploration with real-world applications.

Production of IgG Fusion Proteins Transiently Expressed in Nicotiana benthamiana

We describe here a simple method for expression, extraction, and purification of recombinant human IgG fused to GFP in Nicotiana benthamiana. This protocol can be extended to purification and visualization of numerous proteins that utilize column chromatography. Moreover, the protocol is adaptable to the in-person and virtual college teaching laboratory, providing project-based exploration.

Производство белков IgG Fusion, постоянно выраженных в Никотиана бентамиана

High demand for antibodies as therapeutic interventions for various infectious, metabolic, autoimmune, neoplastic, and other diseases creates a growing ...

Microsurgical Obstruction of Testes Fusion in Spodoptera litura

Instead of using genetic methods like RNA interference (RNAi) and clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated endonuclease Cas9, a physical barrier was microsurgically inserted between the testes of Spodoptera litura to study the impact of this microsurgery on its growth and reproduction. After inserting aluminum foil between the testes, insect molting during metamorphosis proceeded normally. Insect growth and development were not remarkably altered; however, the number of sperm bundles changed if testes fusion was stopped by the microsurgery. These findings imply that blocking testicular fusion can influence male reproduction capability. The method can be further applied to interrupt communication between organs to study the function of specific signaling pathways. Compared to conventional surgery, microsurgery only requires freezing anesthetization, which is preferable to carbon dioxide anesthetization. Microsurgery also minimizes the surgery site area and facilitates wound healing. However, the selection of materials with specific functions needs further investigation. Avoiding tissue injury is crucial when making incisions during the operation.

Microsurgical Obstruction of Testes Fusion in Spodoptera litura

Aluminum foil was microsurgically inserted between the testes of Spodoptera litura to obstruct the fusion of testis. The procedure includes freezing, fixing, disinfection, incision, placing the barrier, suturing, postoperative feeding, and inspection. This approach provides a method to interfere with tissue formation.

Микрохирургическая обструкция слияния яичек в Spodoptera litura

Instead of using genetic methods like RNA interference (RNAi) and clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated ...