저자는 박사 앤 기븐, 박사 크리스틴 맥케이와 모나 동물 서비스 및 생리학 동물 하우스의 모든 직원에게 감사드립니다. 저자 제레미 웨인 스테인과 비디오에 대한 Mcube 팀 및 애니메이션 production.a 감사

<ol><li> 양은 24 시간 동안 금식 물이 광고 libitum가 허용되어 </li><li> 양 아무리입니다. </li><li> Anaestheisa는 thiopentone (20mg/kg)의 정맥 주사에​​ 의해 유발됩니다. </li><li> 예방 정맥 항생제가 주어질 수 있습니다. </li><li> Endotracheal 삽관법는 크기 7-9 수갑에 채워진 endotracheal 튜브를 사용하여 수행됩니다. </li><li> Anaesthesia는 2L 분당의 유량 100 %의 산소, isoflurane, 1-3 %를 maintainted입니다. </li><li> 관찰이 만든 마취의 수준은 지속적으로 모니터링하고 있습니다. </li><li> 이 작업에 필요한 수술 도구는 다음과 같습니다 톱니 포셉, 비 톱니 포셉, Metzenbaum 가위, 메이오 가위, 바늘 홀더, 메스 핸들 (장기 및 단기), 케리슨 rongeurs, 뇌하수체 rongeurs, 공압 수술 드릴, 드릴 비트 및 경비, 신경 갈고리를 (무딘 및 마이크로 훅), 엘리베이터 (즉, 왓슨 샤이엔), 절연 모노 북극과 바이폴라 electrosurgical 포셉, 척추 신체 distractor (예 : 16mm 캐스퍼 핀), 산만 핀 드릴과 드릴 슬리브, 2-0 및 3-0 직각 curettes , 크기 7 10 프랑스어 흡입기와 흡입 튜브, Langenback의 견인기과 자기 유지 경추 견인기. </li><li> 목 지역은 면도와 betadine 세제 솔루션 세탁 있습니다. </li><li> 동물은 다음 운영 테이블에 위로 향한 위치에 배치하고 목을 부드럽게 연장됩니다. </li><li> 피부는 바늘과 X - 레이가 C3 / 4 수준을 확인하기 위해 취해와 함께 표시됩니다. </li><li> 목 그러면 알콜 chlorhexidine, 알콜 요오드와 물 70 % 에탄올을 사용하여 준비합니다. </li><li> fenestrated 광장 드레이프는 수술 사이트와 오버헤드가 테이블에 대한 큰 사각형 드레이프에 사용됩니다. </li><li> 운영 현미경, 또는 루페 확대 및 수리비는 수술을 위해 사용됩니다. </li><li> 절개는 X - 레이를 사용하여 이전에 확인된 사이트에 메스를 사용하여 만든 </li><li> 투자 레이어를 통해 다음 해부 가위를 사용하여 수행되며 비행기 경동맥 칼집과기도와 식도에 측면에 중간 개발했습니다. </li><li> Haemostasis는 투열요법을 사용하여 프로 시저를 통해 유지됩니다 </li><li> 척추 열의가있는되면, 정중선은 palpated되어야하며 치료는 그 철회는 경동맥과기도와 식도에 측면에 중간한지로 이동해야합니다. </li><li> 부상 당했다 주사 바늘을 다음 (이것은 척수를 손상 위험 수있는 실수 발전을 방지) 디스크에 삽입됩니다. 광장 드레이프가 외과 사이트를 커버하는 데 사용됩니다. intraoperative xray은 C3 / 4 디스크 수준을 확인하기 위해 촬영 전에. </li><li> 캐스퍼 산만 핀이 그런 다음 각각 C3와 C4에 삽입됩니다. </li><li> 디스크는 15 메스 블레이드와 베인입니다. </li><li> distractor 그런 다음 삽입 디스크 공간이 부드럽게 떨어져 산만. </li><li> discectomy 그 다음 rongeurs 및 케리슨의 rongeurs를 사용하여 수행됩니다. 자세히 curettes도 도움이 될 수 있습니다. </li><li> endplates 그러면 피가 뼈가 시각까지 자세히 curettes를 사용하여 준비가되어 있습니다. </li><li> 후부 세로 인대는 실험 설계에 따라,이 시점에서 열 수 있습니다. 무딘 후크와 1mm 케리슨이 가장이 프로 시저에 사용됩니다. </li><li> 상처는 그 전에 interbody 케이지 또는 시험 재료의 삽입으로, 정상적인 생리와 irrigated입니다. </li><li> 산만은 광고가 자리에 안전한지 확인하는 검사 장치를 편안하게합니다. </li><li> 산만 핀이 그런 다음 제거하고 뼈를 왁스는 뼈 구멍 haemostasis를 달성하기위한 삽입 수 있습니다. </li><li> longus의 대장균 근육은 Vicryl로 닫힙니다. 계층 클로저는 다음 봉합 제거의 필요성을 피하기 위해 subcuticular 봉합과 선호, 수행됩니다. </li><li> Xylocaine은 수술 현장 주변에 가득 </li><li> 자발 호흡이 발생하고 삼키는의 징후가 분명 때 anaestheitc 가스, 꺼져 있으며, endotracheal 튜브가 제거됩니다. </li><li> 동물은 다음 상수를 관찰 아래 대사 케이지에 복구할 수 있습니다. </li><li> 동물 병리 현상을 방지하기 위해, 그 아래 자리잡고 다리로 직립 위치는 것이 중요합니다. </li><li> 일단 동물은 음식과 물을 reintroduced 수 있습니다 (일반적으로 1시간 이내) 서입니다. </li><li> 닫기 24 시간 동안 수행 감시 1 주일 동안 계속되었습니다. </li><li> 진통제는 수술 다음 3 일 동안 펜타 닐 패치 유지됩니다. </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>

계획 및 앞쪽에 자궁 discectomy과 융합을 수행하는 동안 고려해야 할 다양성의 일부 소스가 있습니다. 따라서 모델의 일관성을 위해, 동물은 동일한 연령과 번식을해야합니다. 우리는 2 살, 잡종 레스터 메리노 양을 선호합니다. 올바르게 수행할 경우, 출혈, 감염, 또는 사망 등과 같은 최소한의 합병증이있을 것입니다. 출혈이 닫기 전에 확인 절차 및 haemosatsis를 통해 제어되어야합니다. 예방 antiobiot...

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		<th>Comment</th>
		</tr>
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		<tbody>
		
<tr>
<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>
<tr>
<td>Chlorehxidine</td>
<td>&nbsp;</td>
<td>Jurox</td>
<td>&nbsp;</td>
<td>40mg/ml Chlorhexidine glucoronate</td>
</tr>
<tr>
<td>PVD Iodine Solution</td>
<td>&nbsp;</td>
<td>Apex</td>
<td>&nbsp;</td>
<td>Povidine Iodine 100mg/ml</td>
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anterior cervical discectomy and fusion in the ovine model

앞쪽에 자궁 경부의 discectomy과 융합 (ACDF)는 보수적인 치료 실패 환자의 자궁 경부 radiculopathy 및 / 또는 myelopathy에 대한 가장 일반적인 수술입니다<sup&gt; 1.5</sup&gt;. 작업 먼저 Cloward에 의해 설명한 이후<sup&gt; 2</sup&gt;와 스미스 앤드 로빈슨<sup&gt; 6</sup&gt; 1958 년에, 기술, 이식 재료와 임플란트의 다양한 개선이되었습니다<sup&gt; 3</sup&gt;. 특히, 선택된 환자에게 이득이 안전 osteoinductive 요원들의 필요가있다. 양의 모델은 인간의 대응, C3 / 4되는 가장 유사한 수준으로 유사한 해부, biomechanical, 뼈 밀도 및 방사능 특성을 가지고 표시되었습니다<sup&gt; 4</sup&gt;. 그것은 따라서 잠복기 연구를 수행할 수있는 이상적인 모델입니다. 특히이 방법은 인간의 척추 수술의 잠재적인 응용 프로그램에 대한 줄기 세포를 포함한 다양한 장치와 biologics을 평가에 관심이 연구자에게 유용할 수 있습니다.

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

이 동영상은 양의 모델 앞쪽에 자궁 discectomy과 융합의 기술을 보여줍니다.

양의 모델의 앞쪽에 자궁암 Discectomy와 퓨전

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 조회수.  Monash University. 이 동영상은 양의 모델 앞쪽에 자궁 discectomy과 융합의 기술을 보여줍니다.

비디오: Anterior Cervical Discectomy and Fusion in the Ovine Model

Method for Measurement of Viral Fusion Kinetics at the Single Particle Level

Membrane fusion is an essential step during entry of enveloped viruses into cells. Conventional fusion assays typically report on a large number of fusion events, making it difficult to quantitatively analyze the sequence of the molecular steps involved. We have developed an in vitro, two-color fluorescence assay to monitor kinetics of single virus particles fusing with a target bilayer on an essentially fluid support.
Influenza viral particles are incubated with a green lipophilic fluorophore to stain the membrane and a red hydrophilic fluorophore to stain the viral interior. We deposit a ganglioside-containing lipid bilayer on the dextran-functionilized glass surface of a flow cell, incubate the viral particles on the planar bilayer and image the fluorescence of a 100 x 100 &mu;m2 area, containing several hundreds of particles, on a CCD camera. By imaging both the red and green fluorescence, we can simultaneously monitor the behavior of the membrane dye (green) and the aqueous content (red) of the particles.
Upon lowering the pH to a value below the fusion pH, the particles will fuse with the membrane. Hemifusion, the merging of the outer leaflet of the viral membrane with the outer leaflet of the target membrane, will be visible as a sudden change in the green fluorescence of a particle. Upon the subsequent fusion of the two remaining distal leaflets a pore will be formed and the red-emitting fluorophore in the viral particle will be released under the target membrane. This event will give rise to a decrease of the red fluorescence of individual particles. Finally, the integrated fluorescence from a pH-sensitive fluorophore that is embedded in the target membrane reports on the exact time of the pH drop.
From the three fluorescence-time traces, all the important events (pH drop, lipid mixing upon hemifusion, content mixing upon pore formation) can now be extracted in a straightforward manner and for every particle individually. By collecting the elapsed times for the various transitions for many individual particles in histograms, we can determine the lifetimes of the corresponding intermediates. Even hidden intermediates that do not have a direct fluorescent observable can be visualized directly from these histograms.

We present an in vitro, two-color fluorescence assay to visualize the fusion of single virus particles with a fluid target bilayer. By labeling viral particles with fluorophores that differentially stain the viral membrane and its interior, we are able to monitor the kinetics of hemifusion and pore formation.

단일 입자 수준에서 바이러스성 퓨전 속도론의 측정 방법

Membrane fusion is an essential step during entry of enveloped viruses into cells. Conventional fusion assays typically report on a large number of fusion ...

연구

생물학

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.

효소 세포 융합 분석을 사용하여 올가미로 인한 막 퓨전 분석

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.

신근 Digitorum의 Longus 근육의 근육 기능의 평가<em&gt; 예 생체</em&gt;와 Tibialis 앞쪽에 근육<em&gt; 현장에서</em&gt; 마우스에

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.

사용<em&gt; 예쁜 꼬마 선충</em&gt; 다세포 생물의 단백질 항상성을 연구하는 모델 시스템으로

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.

총회와 &#39;전단 반지&#39;의 응용 프로그램 : 궤도, 단방향 및 정기 유체 흐름 및 전단 응력에 대한 소설 내피 모델

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.

Murine 골 수 유래 수지상 세포와 대 식 세포에 형광 성 융해 단백질의 표정

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.

레이저 스캐닝 현미경 검사 법에 의해 눈의 앞쪽 챔버에 Thymocytes의 비보에 추적 실시간

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.

효 모에 Polyglutamine 독성 분석 실험을 사용 하 여 퓨전 파트너에 형광 단백질의 영향

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.

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.

니코티아나 벤타미안나에서 과도하게 표현된 IgG 융합 단백질의 생산

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