作者感谢克里斯蒂安Pahrmann（实验室管理器）。

<ol><li>雄性BALB / C小鼠（8-12周）均购自查尔斯河实验室（德国Sulzfeld，）。这些老鼠被安置在常规条件下，美联储标准小鼠的食物和水自由采食。 </li><li> 2％Isofluran是用于麻醉。 </li></ol>供体的制备<ol><li>剃腹毛，消毒面积使用betaisodona。 </li><li>在微观下，执行从喉部水平到胸骨中线的颈椎手术切口。 </li><li>取出皮下脂肪和肩带肌肉得到一个明确的说法气管。 </li><li>任何周围组织解剖的气管，像食道，神经，动脉和结缔组织。 </li><li>删除整个气管分叉喉。 </li><li>用冷盐水冲洗的移植，移植物储存于4 ° C。 </li><li>捐赠者是安乐死颈椎脱位以下收获气管。 </li></ol>收件人：异位移植<ol><li>切口部位周围的大幅度剃腹毛，消毒面积betaisodona（优碘）受酒精使用三次。眼睛应与眼药膏产品润滑，防止角膜干燥。 </li><li>执行中位数的开腹手术，放入无菌手套moistured肠道。 </li><li>仔细蔓延的大网膜。进入中心的移植，并注视单一使用8-0（普理灵，爱惜康，德国）缝合。 </li><li>完全覆盖大网膜移植手术的移植和修复单一缝合8-0（普理灵，爱惜康，德国）。 </li><li>肠子迁移到腹部，并在关闭之前用温水，无菌生理盐水冲洗。 </li><li>关闭2层 - 腹壁的肌肉和皮肤7-0薇乔连续使用7-0普理灵的格局和皮肤层。 </li></ol>收件人：原位移植<ol><li>切口部位周围的大幅度剃腹毛，消毒面积betaisodona（优碘）受酒精使用三次。眼睛应与眼药膏产品润滑，防止角膜干燥。 </li><li>肩带​​肌肉分开，以可视化的全喉复杂。 </li><li>仔细解剖气管周围组织，照顾，保护喉返神经神经。 </li><li>分气管三个环的环状尾鳍。动物通过气管切开保持生理呼吸。 </li><li>确保清洁的收件人，以及在移植气管的边缘。 </li><li>嫁接是收件人气管缺陷和导向，以保持解剖极性之间的中间人。 </li><li>使用8-0（普理灵，爱惜康，德国）吻合的捐助者与远端（纵隔）气管移植。吻合后的执行方面，是在连续运行时装。前方，然后完成中断针的使用。 </li><li>删除从呼吸道分泌物。 </li><li>近端吻合，然后在远端一个相同的方式完成。 </li><li>确保呼吸道的完整性和充足，自主呼吸。 </li><li>搬迁肩带的肌肉和关闭皮下组织和皮肤层使用6-0缝线连续模式（薇乔，爱惜康，德国）。 </li><li>收件人使用注射麻醉，因此将保留通过动物生理呼吸的气管。结合75/1/0.2毫克/公斤，异丙酚medetomidine和芬太尼分别，用于IP麻醉的小鼠。 </li></ol><img src="/files/ftp_upload/1437/1437Fig1.jpg" alt="图1" /> 图1：捐助者的气管。 1A：捐助者的气管就地配制后。 1B：删剪捐助气管。 1C：捐助者气管后explantation。 <img src="/files/ftp_upload/1437/1437Fig2.jpg" alt="图2" /> 图2：异位模型。 2A：移植是在大网膜的中心位置。 2B：移植是用一个单一的缝合两端固定。 2C：移植到大网膜包裹，并用一个单一的缝合固定。 <img src="/files/ftp_upload/1437/1437Fig3.jpg" alt="图3" /> 图3：原位模型。 3A：移植收件人气管缺陷和后壁之间的中间人，是在连续运行时装吻合。 3B：使用单一​​缝合前壁完成。 <img src="/files/ftp_upload/1437/1437Fig4.jpg" alt="图4" /> 图4：切片。 4A：回收后28天在H + E站异位移植气​​管ining（15X）。请注意100％管腔闭塞。 4B：恢复原位移植气管后60天在H + E染色（15X）。最大实现管腔闭塞，约45％。 <table border="1"><tbody><tr><td></td><td> 异位气管移植模型 </td><td> 原位气管移植模型 </td></tr><tr><td> 优势 </td><td> +容易执行 +完整的气道阻塞的管腔闭塞后28天 +无由OAD的动物的身体感情 </td><td> +物理通风的嫁接 +吸入药品监督管理局 +强免疫反应，如同种异体IgM抗体生产 +胸椎的生理环境 +气管气管吻合模仿临床</td></tr><tr><td> 缺点 </td><td> - 没有移植气管的通风 - 没有评价吸入病原体的可能 - 抑制纤毛清除率和保留分泌物 - 腹膜微环境，而不是胸环境 </td><td> - 手术必要的培训 - 管腔闭塞管腔闭塞的应用程序。经过60天的45％ - 动物可能发展的OAD的症状 </td></tr></tbody></table> 表1：异位和原位气管移植的优点和缺点。

<ol>
	<li>Adams, B., Berry, G., Huang, X., Shorthouse, R., Brazelton, T., Morris, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunosuppressive+therapies+for+the+prevention+and+treatment+of+obliterative+airway+disease+in+heterotopic+rat+trachea+allografts.">Immunosuppressive therapies for the prevention and treatment of obliterative airway disease in heterotopic rat trachea allografts.</a> Transplantation. 69, 2260-2266 .</li><li>Adams, B., Brazelton, T., Berry, G., Morris, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+respiratory+epithelium+in+a+rat+model+of+obliterative+airway+disease.">The role of respiratory epithelium in a rat model of obliterative airway disease.</a> Transplantation. 69, 661-665 .</li><li>Deuse, T., Schrepfer, S., Reichenspurner, H., Hoyt, G., Fischbein, M., Robbins, R., Pelletier, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Techniques+for+experimental+heterotopic+and+orthotopic+tracheal+transplantations+&amp;#x2013;+when+to+use+which+model;+Transplant+Immunology+17.">Techniques for experimental heterotopic and orthotopic tracheal transplantations &amp;#x2013; when to use which model; Transplant Immunology 17.</a> , 255-261 (2007).</li><li>Hele, D., Yacoub, M., Belvisi, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+heterotopic+tracheal+allograft+as+an+animal+model+of+obliterative+bronchiolitis.">The heterotopic tracheal allograft as an animal model of obliterative bronchiolitis.</a> Respiratory Research. 2, 169-183 .</li><li>Hertz, M., Jessurun, J., King, M., Savic, S., Murray, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reproduction+of+the+obliterative+bronchiolitis+lesion+after+heterotopic+transplantation+of+mouse+airways.">Reproduction of the obliterative bronchiolitis lesion after heterotopic transplantation of mouse airways.</a> American Journal of Pathology. 142, (1993).</li><li>McDyer, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+and+murine+obliterative+bronchiolitis+in+transplant.">Human and murine obliterative bronchiolitis in transplant.</a> Proceedings of the American Thoracic Society. 4, 37-43 .</li><li>Schrepfer, S., Deuse, T., Hoyt, G., Sheikh, A., Hoffmann, J., Reichenspurner, H., Robbins, R., Pelletier, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Experimental+orthotopic+tracheal+transplantation:+the+stanford+technique.">Experimental orthotopic tracheal transplantation: the stanford technique.</a> Microsurgery. 27, 187-189 (2007).</li></ol>

宋佳Schrepfer已收到由德意志研究联合会（DFG）（SCHR992/3-1）的研究补助金。] 所有的动物都收到符合实验动物保健的原则“的人文关怀，对动物的实验是在由国家医学研究和指南学会出版的”实验动物的关心和使用规定的准则和法规美国国立卫生研究院（美国国立卫生出版物85-23研究院，1985年修订）。 所有动物均来自查尔斯河实验室（德国Sulzfeld，），并保持在汉堡大学医院的Eppendorf的动物保健设施。动物标准的议员和水自由采食。

小鼠可在不同的transgeneic和淘汰赛模式，因此适合研究的OAD（4）有关的机械问题。 在此视频中显示的气管移植两种型号，可作为可靠的模型研究OAD的发展。 然而，每一个模型演示的优势和局限。 异位气管移植是很容易执行，并不需要特殊的手术训练（3，5） 。异位移植后，管腔闭塞将出现快速，完整的气道阻塞出现后28天（3，4，6）。动物身体的影响O...

heterotopic and orthotopic tracheal transplantation in mice used as models to study the development of  obliterative airway disease

闭塞性气道疾病（OAD）主要的并发症是肺移植后，限制长期生存（1-7）。 为了研究的病理生理，治疗和预防OAD的不同的动物模型，在啮齿类动物的气管移植已开发（1-7）。在这里，我们使用两个气管移植，异位和原位模型建立的模型，并展示自己的优点和局限性。 对于异位模型，捐赠者的气管进入收件人的大网膜包裹，而捐助者的气管原位模型是在结束对端吻合吻合。 在这两种模式的闭塞性病变的组织学类似临床的OAD的发展已经证明（1-7）。 本视频演示了如何同时执行，异位以及小鼠原位气管移植技术，比较OAD的发展过程中都使用的组织学模型。

Watch this Scientific Journal Video about Heterotopic and Orthotopic Tracheal Transplantation in Mice used as Models to Study the Development of  Obliterative Airway Disease at JoVE.com

Heterotopic and Orthotopic Tracheal Transplantation in Mice used as Models to Study the Development of  Obliterative Airway Disease

该视频显示和比较两个实验模型来研究发展的闭塞性气道疾病（OAD），在小鼠体内的异位和原位气管移植模型。

异位和原位气管移植小鼠模型研究闭塞性气道疾病的发展

Obliterative airway disease (OAD) is the major complication after lung transplantations that limits long term survival (1-7).
To study the ...

heterotopic-orthotopic-tracheal-transplantation-mice-used-as-models

Research

JoVE Journal

Biology

13.8K 次观看.  University Heart Center Hamburg. 该视频显示和比较两个实验模型来研究发展的闭塞性气道疾病（OAD），在小鼠体内的异位和原位气管移植模型。

视频: Heterotopic and Orthotopic Tracheal Transplantation in Mice used as Models to Study the Development of  Obliterative Airway Disease

Testing Visual Sensitivity to the Speed and Direction of Motion in Lizards

Testing visual sensitivity in any species provides basic information regarding behaviour, evolution, and ecology. However, testing specific features of the visual system provide more empirical evidence for functional applications. Investigation into the sensory system provides information about the sensory capacity, learning and memory ability, and establishes known baseline behaviour in which to gauge deviations (Burghardt, 1977). However, unlike mammalian or avian systems, testing for learning and memory in a reptile species is difficult. Furthermore, using an operant paradigm as a psychophysical measure of sensory ability is likewise as difficult. Historically, reptilian species have responded poorly to conditioning trials because of issues related to motivation, physiology, metabolism, and basic biological characteristics. Here, I demonstrate an operant paradigm used a novel model lizard species, the Jacky dragon (Amphibolurus muricatus) and describe how to test peripheral sensitivity to salient speed and motion characteristics. This method uses an innovative approach to assessing learning and sensory capacity in lizards. I employ the use of random-dot kinematograms (RDKs) to measure sensitivity to speed, and manipulate the level of signal strength by changing the proportion of dots moving in a coherent direction. RDKs do not represent a biologically meaningful stimulus, engages the visual system, and is a classic psychophysical tool used to measure sensitivity in humans and other animals. Here, RDKs are displayed to lizards using three video playback systems. Lizards are to select the direction (left or right) in which they perceive dots to be moving. Selection of the appropriate direction is reinforced by biologically important prey stimuli, simulated by computer-animated invertebrates.

Testing visual sensitivity in lizards using an operant conditioning paradigm that employs video playback of random-dot kinematograms and computer-generated invertebrates.

测试的速度和运动方向的视觉灵敏度在蜥蜴

Testing visual sensitivity in any species provides basic information regarding behaviour, evolution, and ecology. However, testing specific features of ...

科研

生物学

Heterotopic Heart Transplantation in Mice

The mouse heterotopic heart transplantation has been used widely since it was introduced by Drs. Corry and Russell in 1973. It is particularly valuable for studying rejection and immune response now that newer transgenic and gene knockout mice are available, and a large number of immunologic reagents have been developed. The heart transplant model is less stringent than the skin transplant models, although technically more challenging. We have developed a modified technique and have completed over 1000 successful cases of heterotopic heart transplantation in mice. When making anastomosis of the ascending aorta and abdominal aorta, two stay sutures are placed at the proximal and distal apexes of recipient abdominal aorta with the donor s ascending aorta, then using 11-0 suture for anastomosis on both side of aorta with continuing sutures. The stay sutures make the anastomosis easier and 11-0 is an ideal suture size to avoid bleeding and thrombosis.
When making anastomosis of pulmonary artery and inferior vena cava, two stay sutures are made at the proximal apex and distal apex of the recipient s inferior vena cava with the donor s pulmonary artery. The left wall of the inferior vena cava and donor s pulmonary artery is closed with continuing sutures in the inside of the inferior vena cava after, one knot with the proximal apex stay suture the right wall of the inferior vena cava and the donor s pulmonary artery are closed with continuing sutures outside the inferior vena cave with 10-0 sutures. This method is easier to perform because anastomosis is made just on the one side of the inferior vena cava and 10-0 sutures is the right size to avoid bleeding and thrombosis. In this article, we provide details of the technique to supplement the video.

The mouse heterotopic heart transplantation model has been proven by many investigators to be an important method for studying mechanisms of rejection and immune response. However, the techniques involved are still challenging. By modifying standard techniques we have had success with more than 1000 transplants.

小鼠异位心脏移植

The mouse heterotopic heart transplantation has been used widely since it was introduced by Drs. Corry and Russell in 1973. It is particularly valuable ...

Quantification of &gamma;H2AX Foci in Response to Ionising Radiation

DNA double-strand breaks (DSBs), which are induced by either endogenous metabolic processes or by exogenous sources, are one of the most critical DNA lesions with respect to survival and preservation of genomic integrity. An early response to the induction of DSBs is phosphorylation of the H2A histone variant, H2AX, at the serine-139 residue, in the highly conserved C-terminal SQEY motif, forming &gamma;H2AX1. Following induction of DSBs, H2AX is rapidly phosphorylated by the phosphatidyl-inosito 3-kinase (PIKK) family of proteins, ataxia telangiectasia mutated (ATM), DNA-protein kinase catalytic subunit and ATM and RAD3-related (ATR)2. Typically, only a few base-pairs (bp) are implicated in a DSB, however, there is significant signal amplification, given the importance of chromatin modifications in DNA damage signalling and repair. Phosphorylation of H2AX mediated predominantly by ATM spreads to adjacent areas of chromatin, affecting approximately 0.03% of total cellular H2AX per DSB2,3. This corresponds to phosphorylation of approximately 2000 H2AX molecules spanning ~2 Mbp regions of chromatin surrounding the site of the DSB and results in the formation of discrete &gamma;H2AX foci which can be easily visualized and quantitated by immunofluorescence microscopy2. The loss of &gamma;H2AX at DSB reflects repair, however, there is some controversy as to what defines complete repair of DSBs; it has been proposed that rejoining of both strands of DNA is adequate however, it has also been suggested that re-instatement of the original chromatin state of compaction is necessary4-8. The disappearence of &gamma;H2AX involves at least in part, dephosphorylation by phosphatases, phosphatase 2A and phosphatase 4C5,6. Further, removal of &gamma;H2AX by redistribution involving histone exchange with H2A.Z has been implicated7,8. Importantly, the quantitative analysis of &gamma;H2AX foci has led to a wide range of applications in medical and nuclear research. Here, we demonstrate the most commonly used immunofluorescence method for evaluation of initial DNA damage by detection and quantitation of &gamma;H2AX foci in &gamma;-irradiated adherent human keratinocytes9.

Quantification of &amp;#947;H2AX Foci in Response to Ionising Radiation

Quantification of DNA double-strand streaks using &gamma;H2AX formation as a molecular marker has become an invaluable tool in radiation biology. Here we demonstrate the use of an immunofluorescence assay for quantification of &gamma;H2AX foci after exposure of cells to radiation.

电离辐射的反应定量的γH2AX灶

DNA double-strand breaks (DSBs), which are induced by either endogenous metabolic processes or by exogenous sources, are one of the most critical DNA ...

Manual Restraint and Common Compound Administration Routes in Mice and Rats

Being able to safely and effectively restrain mice and rats is an important part of conducting research. Working confidently and humanely with mice and rats requires a basic competency in handling and restraint methods. This article will present the basic principles required to safely handle animals. One-handed, two-handed, and restraint with specially designed restraint objects will be illustrated. Often, another part of the research or testing use of animals is the effective administration of compounds to mice and rats. Although there are a large number of possible administration routes (limited only by the size and organs of the animal), most are not used regularly in research. This video will illustrate several of the more common routes, including intravenous, intramuscular, subcutaneous, and oral gavage. The goal of this article is to expose a viewer unfamiliar with these techniques to basic restraint and substance administration routes. This video does not replace required hands-on training at your facility, but is meant to augment and supplement that training.

Working safely and humanely with research rodents requires a core competency in handling and restraint methods. This article will present the basic principles required to safely handle and effectively administer compounds to mice and rats.

手动约束和常见的化合物在小鼠和大鼠的给药途径

Being able to safely and effectively restrain mice and rats is an important part of conducting research.  Working confidently and humanely with mice and ...

Evaluation of Mammary Gland Development and Function in Mouse Models

The human mammary gland is composed of 15-20 lobes that secrete milk into a branching duct system opening at the nipple. Those lobes are themselves composed of a number of terminal duct lobular units made of secretory alveoli and converging ducts1. In mice, a similar architecture is observed at pregnancy in which ducts and alveoli are interspersed within the connective tissue stroma. The mouse mammary gland epithelium is a tree like system of ducts composed of two layers of cells, an inner layer of luminal cells surrounded by an outer layer of myoepithelial cells denoted by the confines of a basement membrane2. At birth, only a rudimental ductal tree is present, composed of a primary duct and 15-20 branches. Branch elongation and amplification start at the beginning of puberty, around 4 weeks old, under the influence of hormones3,4,5. At 10 weeks, most of the stroma is invaded by a complex system of ducts that will undergo cycles of branching and regression in each estrous cycle until pregnancy2. At the onset of pregnancy, a second phase of development begins, with the proliferation and differentiation of the epithelium to form grape-shaped milk secretory structures called alveoli6,7. Following parturition and throughout lactation, milk is produced by luminal secretory cells and stored within the lumen of alveoli. Oxytocin release, stimulated by a neural reflex induced by suckling of pups, induces synchronized contractions of the myoepithelial cells around the alveoli and along the ducts, allowing milk to be transported through the ducts to the nipple where it becomes available to the pups 8. Mammary gland development, differentiation and function are tightly orchestrated and require, not only interactions between the stroma and the epithelium, but also between myoepithelial and luminal cells within the epithelium9,10,11. Thereby, mutations in many genes implicated in these interactions may impair either ductal elongation during puberty or alveoli formation during early pregnancy, differentiation during late pregnancy and secretory activation leading to lactation12,13. In this article, we describe how to dissect mouse mammary glands and assess their development using whole mounts. We also demonstrate how to evaluate myoepithelial contractions and milk ejection using an ex-vivo oxytocin-based functional assay. The effect of a gene mutation on mammary gland development and function can thus be determined in situ by performing these two techniques in mutant and wild-type control mice.

This method describes how to dissect and assess mammary gland development and function from mice. Excised mammary glands are assessed for the degree of development using whole mount while milk ejection is evaluated using an oxytocin-based myoepithelial cell contraction assay.

乳腺的发育和功能在小鼠模型的评价

The human mammary gland is composed of 15-20 lobes that secrete milk into a branching duct system opening at the nipple. Those lobes are themselves ...

Diagnostic Necropsy and Selected Tissue and Sample Collection in Rats and Mice

There are multiple sample types that may be collected from a euthanized animal in order to help diagnose or discover infectious agents in an animal colony. Proper collection of tissues for further histological processing can impact the quality of testing results. This article describes the conduct of a basic gross examination including identification of heart, liver, lungs, kidneys, and spleen, as well as how to collect those organs. Additionally four of the more difficult tissue/sample collection techniques are demonstrated. Lung collection and perfusion can be particularly challenging as the tissue needs to be properly inflated with a fixative in order for inside of the tissue to fix properly and to enable thorough histologic evaluation. This article demonstrates the step by step technique to remove the lung and inflate it with fixative in order to achieve optimal fixation of the tissue within 24 hours. Brain collection can be similarly challenging as the tissue is soft and easily damaged. This article demonstrates the step by step technique to expose and remove the brain from the skull with minimal damage to the tissue. The mesenteric lymph node is a good sample type in which to detect many common infectious agents as enteric viruses persist longer in the lymph node than they are shed in feces. This article demonstrates the step by step procedure for locating and aseptically removing the mesenteric lymph node. Finally, identification of infectious agents of the respiratory tract may be performed by bacterial culture or PCR testing of nasal and/or bronchial fluid aspirates taken at necropsy. This procedure describes obtaining and preparing the respiratory aspirate sample for bacterial culture and PCR testing.

This article describes the procedures for conducting a basic postmortem examination of a mouse or rat, and the collection of basic organs, as well as more challenging sample types from for histological, microbiological, and PCR evaluation.

剖检诊断，并选择在大鼠和小鼠的组织和样品收集

There are multiple sample types that may be collected from a euthanized animal in order to help diagnose or discover infectious agents in an animal ...

Do-It-Yourself Device for Recovery of Cryopreserved Samples Accidentally Dropped into Cryogenic Storage Tanks

Liquid nitrogen is colorless, odorless, extremely cold (-196 &deg;C) liquid kept under pressure. It is commonly used as a cryogenic fluid for long term storage of biological materials such as blood, cells and tissues 1,2. The cryogenic nature of liquid nitrogen, while ideal for sample preservation, can cause rapid freezing of live tissues on contact - known as 'cryogenic burn'2, which may lead to severe frostbite in persons closely involved in storage and retrieval of samples from Dewars. Additionally, as liquid nitrogen evaporates it reduces the oxygen concentration in the air and might cause asphyxia, especially in confined spaces2.
In laboratories, biological samples are often stored in cryovials or cryoboxes stacked in stainless steel racks within the Dewar tanks1. These storage racks are provided with a long shaft to prevent boxes from slipping out from the racks and into the bottom of Dewars during routine handling. All too often, however, boxes or vials with precious samples slip out and sink to the bottom of liquid nitrogen filled tank. In such cases, samples could be tediously retrieved after transferring the liquid nitrogen into a spare container or discarding it. The boxes and vials can then be relatively safely recovered from emptied Dewar. However, the cryogenic nature of liquid nitrogen and its expansion rate makes sunken sample retrieval hazardous. It is commonly recommended by Safety Offices that sample retrieval be never carried out by a single person. Another alternative is to use commercially available cool grabbers or tongs to pull out the vials3. However, limited visibility within the dark liquid filled Dewars poses a major limitation in their use.
In this article, we describe the construction of a Cryotolerant DIY retrieval device, which makes sample retrieval from Dewar containing cryogenic fluids both safe and easy.

Here we present a low cost, durable cryotolerant device for sample retrieval from Dewar tanks filled with liquid nitrogen. The ease of construction and modular design of the device makes the process of sample retrieval from cryogenic tanks safe and easy.

做自己动手恢复冻存样品的设备不慎掉落到低温储罐

Liquid nitrogen is colorless, odorless, extremely cold (-196 &deg;C) liquid kept under pressure. It is commonly used as a cryogenic fluid for long term ...

Using SecM Arrest Sequence as a Tool to Isolate Ribosome Bound Polypeptides

Extensive research has provided ample evidences suggesting that protein folding in the cell is a co-translational process1-5. However, the exact pathway that polypeptide chain follows during co-translational folding to achieve its functional form is still an enigma. In order to understand this process and to determine the exact conformation of the co-translational folding intermediates, it is essential to develop techniques that allow the isolation of RNCs carrying nascent chains of predetermined sizes to allow their further structural analysis.
SecM (secretion monitor) is a 170 amino acid E. coli protein that regulates expression of the downstream SecA (secretion driving) ATPase in the secM-secA operon6. Nakatogawa and Ito originally found that a 17 amino acid long sequence (150-FSTPVWISQAQGIRAGP-166) in the C-terminal region of the SecM protein is sufficient and necessary to cause stalling of SecM elongation at Gly165, thereby producing peptidyl-glycyl-tRNA stably bound to the ribosomal P-site7-9. More importantly, it was found that this 17 amino acid long sequence can be fused to the C-terminus of virtually any full-length and/or truncated protein thus allowing the production of RNCs carrying nascent chains of predetermined sizes7. Thus, when fused or inserted into the target protein, SecM stalling sequence produces arrest of the polypeptide chain elongation and generates stable RNCs both in vivo in E. coli cells and in vitro in a cell-free system. Sucrose gradient centrifugation is further utilized to isolate RNCs.
The isolated RNCs can be used to analyze structural and functional features of the co-translational folding intermediates. Recently, this technique has been successfully used to gain insights into the structure of several ribosome bound nascent chains10,11. Here we describe the isolation of bovine Gamma-B Crystallin RNCs fused to SecM and generated in an in vitro translation system.

We describe here a technique that is now routinely used to isolate stably bound ribosome nascent chain complexes (RNCs). This technique takes advantage of the discovery that a 17 amino acid long SecM "arrest sequence" can halt translation elongation in a prokaryotic (E. coli) system, when inserted into (or fused to the C-terminus) of virtually any protein.

作为一种工具来隔离核糖体的束缚多肽SECM逮捕序列

Extensive research has provided ample evidences suggesting that protein folding in the cell is a co-translational process1-5. However, the exact pathway ...

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 ...

Tissue Triage and Freezing for Models of Skeletal Muscle Disease

Skeletal muscle is a unique tissue because of its structure and function, which requires specific protocols for tissue collection to obtain optimal results from functional, cellular, molecular, and pathological evaluations. Due to the subtlety of some pathological abnormalities seen in congenital muscle disorders and the potential for fixation to interfere with the recognition of these features, pathological evaluation of frozen muscle is preferable to fixed muscle when evaluating skeletal muscle for congenital muscle disease. Additionally, the potential to produce severe freezing artifacts in muscle requires specific precautions when freezing skeletal muscle for histological examination that are not commonly used when freezing other tissues. This manuscript describes a protocol for rapid freezing of skeletal muscle using isopentane (2-methylbutane) cooled with liquid nitrogen to preserve optimal skeletal muscle morphology. This procedure is also effective for freezing tissue intended for genetic or protein expression studies. Furthermore, we have integrated our freezing protocol into a broader procedure that also describes preferred methods for the short term triage of tissue for (1) single fiber functional studies and (2) myoblast cell culture, with a focus on the minimum effort necessary to collect tissue and transport it to specialized research or reference labs to complete these studies. Overall, this manuscript provides an outline of how fresh tissue can be effectively distributed for a variety of phenotypic studies and thereby provides standard operating procedures (SOPs) for pathological studies related to congenital muscle disease.

The analysis of skeletal muscle tissues to determine structural, functional, and biochemical properties is greatly facilitated by appropriate preparation. This protocol describes appropriate methods to prepare skeletal muscle tissue for a broad range of phenotyping studies.

组织分流和冻结的骨骼肌疾病模型

Skeletal muscle is a unique tissue because of its structure and function, which requires specific protocols for tissue collection to obtain optimal ...