著者らは、クリスティPahrmannを（Lab Managerの）感謝。

<ol><li>雄BALB / cマウス（8-12週）はチャールズリバー研究所（Sulzfeld、ドイツ）から購入されています。マウスは通常の条件、飼育、標準のマウスの食物と水を自由の下に収容されています。 </li><li> 2％のIsofluranは麻酔に使用されます。 </li></ol>ドナーの調製<ol><li>腹部の毛を剃るとbetaisodonaを使用して領域を消毒する。 </li><li>顕微鏡視野の下で、喉頭のレベルから胸骨に正中頸部切開を行います。 </li><li>気管のクリアな視界を得るために皮下脂肪とストラップの筋肉を取り外します。 </li><li>食道、神経、動脈、及び結合組織のように、あらゆる周囲の組織から気管を細かく分析。 </li><li>全体気管を（喉頭から分岐まで）を取り外します。 </li><li>冷たい生理食塩水で移植をフラッシュし、4でグラフト℃にて保存してください。 </li><li>ドナーは、気管の収穫後の頚椎脱臼により安楽死されています。 </li></ol> RECIPIENT：異種移植<ol><li>切開部位周辺の広いマージンで腹部の毛を剃るとbetaisodona（betadine）を用いて3回はアルコールが続くエリアを消毒する。目が乾燥から角膜を防ぐために、眼軟膏の製品で潤滑する必要があります。 </li><li>中央開腹を行い、滅菌、moistured手袋の中に腸を置きます。 </li><li>慎重に大網を広げる。中央に移植片を置き、8から0（Prolene、エチコン、ドイツ）を使用して単一の縫合糸でそれを凝視する。 </li><li>完全に大網を移植をカバーし、一つの縫合8から0（Prolene、エチコン、ドイツ）と移植片を修正。 </li><li>腹部に戻して腸を再配置し、閉鎖する前に暖かい、滅菌生理食塩水で洗い流してください。 </li><li> 2層でクローズアップ - 筋肉と皮膚のための7から0 Vicrylための7から0 Proleneを使用して連続的なパターンと腹壁と皮膚の層。 </li></ol> RECIPIENT：同所移植<ol><li>切開部位周辺の広いマージンで腹部の毛を剃るとbetaisodona（betadine）を用いて3回はアルコールが続くエリアを消毒する。目が乾燥から角膜を防ぐために、眼軟膏の製品で潤滑する必要があります。 </li><li>全体喉頭複雑を視覚化するためにストラップの筋肉を分ける。 </li><li>慎重に、周囲の組織から気管を解剖反回神経を維持するように注意してください。 </li><li>輪状軟骨から尾側気管3つのリングを分ける。動物は気管切開を介して生理的な呼吸を維持します。 </li><li>受信者のほか、移植片のクリーン気管エッジを確認してください。 </li><li>移植片は、解剖学的極性を維持するために欠陥気管と志向受信者との間に介在している。 </li><li>吻合する遠位（縦隔）気管とドナーの移植片は（Prolene、エチコン、ドイツ）8から0を使用する。吻合の後面は、連続運転方式で実行されます。前面は中断ステッチを使用して完了します。 </li><li>気道からの分泌物を除去する。 </li><li>近位吻合は、遠位のものと同じ方法で完了します。 </li><li>気道と十分な、自発呼吸の整合性を確認してください。 </li><li>ストラップの筋肉を再配置し、連続的なパターンで6から0縫合（Vicryl、エチコン、ドイツ）を使用して皮下組織と皮膚の層を閉じます。 </li><li>受信者のために注射麻酔を使用して、そのため動物は気管を介して生理的な呼吸を保持します。それぞれプロポフォール、メデトミジンとフェンタニルの75/1/0.2 mg / kgの組み合わせは、、マウスでIP麻酔に使用されます。 </li></ol><img src="/files/ftp_upload/1437/1437Fig1.jpg" alt="図1" /> 図1：ドナー気管。 1A：調製後のin situドナー気管。 1B：摘出ドナー気管。 1C：植後のドナー気管。 <img src="/files/ftp_upload/1437/1437Fig2.jpg" alt="図2" /> 図2：異所性モデル。 2A：移植片は、大網の中心に位置しています。 2B：移植片は、単一の縫合糸の両端に固定されている。 2C：移植片を大網に包まれ、一針の縫合で固定されている。 <img src="/files/ftp_upload/1437/1437Fig3.jpg" alt="図3" /> 図3：同所性モデル。 3A：移植片がレシピエント気管欠陥と後壁との間に介在され、連続的な実行方法で吻合される。 3B：前壁が単一の縫合糸を使用して完了する。 <img src="/files/ftp_upload/1437/1437Fig4.jpg" alt="図4" /> 図4：組織学。 4A：H + E STAで28日後に回収された異所性移植気管ining（15倍）。 100パーセント内腔の閉塞に注意してください。 4Bは：H + E染色（15倍）で60日後に同所性移植気管を回復した。最大達成管腔閉塞は約45％です。 <table border="1"><tbody><tr><td></td><td> 異所性気管移植モデル </td><td> 同所性気管移植モデル </td></tr><tr><td> 利点 </td><td>実行するには+が簡単 28日後に完全な気道閉塞と+腔内閉塞 OADによる動物の+は物理的な愛情なし </td><td>移植片の+物理換気 可能+吸入薬の投与 このようなalloreactive IgM抗体の産生など、+強力な免疫反応 +生理胸部環境 +気管 - 気管吻合は、臨床の現場を模したもの</td></tr><tr><td> 欠点 </td><td> - 移植気管のない換気 - 吸入病原体のない評価が可能 - 粘液線毛クリアランスや保持分泌の阻害 - 腹膜微小環境の代わりに胸部環境 </td><td> - 必要な外科研修 - 管腔の閉塞アプリと腔内閉塞。 60日後の45パーセント - 動物は、OADの症状を発症することが </td></tr></tbody></table> 表1：異所性と同所性気管移植の長所と短所。

<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>

ソーニャSchrepferは、ドイツ学術振興協会（DFG）（SCHR992/3-1）から研究助成を受けています。] すべての動物は、動物実験は医学研究とによって公開実験動物の管理と利用のためのガイドのための国立協会が定めるガイドラインおよび規制に準拠して行われた、実験動物のケアの原則に準拠して人間のケアを受け国立衛生研究所（厚生出版物85から23の国立研究所、1985年改訂）。 すべての動物は、チャールズリバーラボラトリーズ（Sulzfeld、ドイツ）から入手し、大学病院ハンブルクエッペンドルフの動物医療施設で維持した。動物は、標準の固形飼料と水を自由に摂取を受けた。

マウスは別のtransgeneicとノックアウトモデルで提供され、OAD（4）に関連する機械的な問題を研究することが適しています。 このビデオに示されている気管移植の両方のモデルは、OADの開発を研究するための信頼性の高いモデルとして使用することができます。 しかし、各モデルには長所と限界を示しています。 異所性気管移植が容易に実行できま...

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

閉塞性気道疾患（OAD）が長期生存（1-7）を制限する肺移植後の主要な合併症である。 病態生理学を研究するために、げっ歯類における気管移植のOAD、さまざまな動物モデルの治療と予防は、（1-7）が開発されている。ここで、我々は気管移植、異所性と同所性モデルの確立された2つのモデルを使用し、その利点と限界を示しています。 異所性モデルの場合は、気管が受信者の大網にラップされているドナーは、ドナーに対し、気管は、同所性モデルにおけるエンドツーエンドの吻合によって吻合される。 両方のモデルでは、臨床OADに似た組織学的閉塞性病変の発生は、（1-7）実証されている。 このビデオは両方を実行する方法を示し、異所性だけでなく、同​​所性気管移植のテクニックマウスで、とは、組織を使用して、両方のモデルでOAD開発の時間経過を比較します。

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

このビデオでは、マウスでは閉塞性気道疾患（OAD）の開発を研究するには、2つの実験モデル、異所および同所性気管移植モデルを示し、比較します。

閉塞性気道疾患の発生研究のモデルとして使用されるマウスにおける異所性と同所性気管移植

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 ビュー.  University Heart Center Hamburg. このビデオでは、マウスでは閉塞性気道疾患（OAD）の開発を研究するには、2つの実験モデル、異所および同所性気管移植モデルを示し、比較します。

ビデオ: 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.

トカゲの運動の速度と方向の視覚感度のテスト

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

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.

マウスにおける異所心臓移植

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.

電離放射線への応答でγH2AX病巣の定量化

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.

マウスとラットの拘束マニュアルと共通の複合投与経路

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.

マウスモデルにおける乳腺の発達と機能の評価

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.

診断剖検及びラット及びマウスで選択された組織とサンプル集

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.

凍結保存サンプルの回復のための日曜大工のデバイスが誤って低温貯蔵タンクに落とし

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.

リボソーム結合したポリペプチドを単離するためのツールと​​してSECM逮捕シーケンスを使用して、

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.

組織トリアージや骨格筋疾患のモデルのために凍結

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