<ol><li> IACUC承認された手順を使用して、イソフルランによるマウス（誘導は4％、手術のための1.5から2パーセント）を麻酔。尾及び/またはTOEのつまみが動物を完全に鎮静されていることを確認するために使用されていることが重要です。 </li><li>げっ歯類のトリマを使用して、上の目に首の後ろから髪を剃る。 </li><li>手術の水再循環ブランケットを介して、定位固定フレームにマウスを置きます。しっかりと耳を棒で頭を固定します。 </li><li>乾燥から動物の目を防ぐために、眼軟膏を適用します。 </li><li>それぞれ、脳および/または炎症反応の腫れを防ぐために、皮下、管理デキサメタゾン（0.2 mg / kg体重）とカルプロフェン（5 mg / kg体重）。 </li><li>手術を始める前に、70％アルコールとbetadineの三交互にスワイプで皮膚を拭いて動作領域を滅菌する。 </li><li>すべての手術器具は、ガラスビーズ滅菌器を使用して事前に滅菌されています。エタノールで滅菌されたはさみを使用して、2つの吻側方向へのカット、ほとんどまぶたに達し、二つの斜めカットに続くすべての頭部のベースに沿って水平方向のカット、で始まる、頭蓋骨の上に皮膚を取り除くそれは、正中線に収束する。 </li><li>リドカイン+エピネフリンの溶液の液滴は、過剰な出血や痛みを避けるために、骨膜の上に、この時点で適用されます。メスで、頭蓋骨の縁に骨膜を引っ込める。また、軽く首の後ろの筋肉を引っ込める。 </li><li>穏やかに乾燥した表面を作成するためにメスで頭蓋骨の全体の露出面積を削る。後で適用されたとき、それは接着剤が良く付着できるようになりますので、これは非常に重要です。 </li><li>イメージングサイトが選択されると、一つは頭蓋のウィンドウを作成する準備ができています。最初に、静かに空気歯科用ドリルで直径約4 mmの円を&quot;描く&quot;。 </li><li>わずかな掘削した後、頭蓋骨の表面に再びリドカイン+エピネフリンのソリューションを適用する。骨の非常に薄い層が残っている掘削を停止します。それは道を与えるか感じるように開頭の中央に軽く押すことで、一つは通常、この段階に到達したことを知ることができます。 </li><li>骨の海綿構造 - - 骨梁の生理食塩水と取る利点の低下の下で非常に細い先端の鉗子で頭蓋骨から開頭を持ち上げて外します。それは頭蓋骨を持ち上げと硬膜の出血を防ぐのを助けるので生理食塩水は、重要です。 </li><li>以前に頭蓋骨が削除されるとき、時々発生した小さな出血を止めるために硬膜に生理食塩水に浸したされたGelfoamを適用します。 </li><li>硬膜の表面を乾燥や出血がないことを確認した後、ゆっくりと硬膜の上に滅菌5 mmのガラス製カバースリップを置く。 （ 注 ：他のグループも、硬膜上に低融点アガロース（1.2％）の低下を置き、寒天の上にカバースリップを置く）。 </li><li>頭蓋骨上の反対側の半球にcyanocrylateベースの接着剤の滴を適用します。ガラスの下に置くように注意しながらしながら針の助けを借りて、ゆっくりとすべてのウィンドウの周囲に接着剤を適用する。接着剤は、現在頭蓋骨の表面全体に薄い層に適用することができます。 </li><li>接着剤が乾燥したら、それを確保するため、また、カバースリップの小さな縁をカバーする、頭蓋骨の表面全体に歯のアクリルをミックスし、それを適用します。 </li><li>カバースリップを確保した後、歯科用アクリルを使用してウィンドウの周りに小さな井戸を行います。また、歯科アクリルのチタン棒を埋め込みます。このバーは、後のイメージングのための顕微鏡のステージにしっかりとマウスを接続するために使用されます。それは頭蓋窓と平行になるように、バーが水平であることを確認することが重要です。バーの下に一枚の紙を置くと、アクリルが硬化しながらバーのレベルを維持できるようにすることができます。 </li><li>歯科アクリルが10分間（強化）硬化され、その時点でチタン棒を所定の位置に固定されています。それが回復するまでは暖かいケージに動物を置きます。 </li><li>麻酔から回復した後、動物は、同じ日に結像することができます。 </li></ol>

<ol>
	<li>Svoboda, K., Denk, W., Kleinfeld, D., Tank, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=+In+vivo+dendritic+calcium+dynamics+in+neocortical+pyramidal+neurons."> In vivo dendritic calcium dynamics in neocortical pyramidal neurons.</a> Nature. 85, 161-165 (1997).</li><li>Lendvai, B., Stern, E. A., Chen, B., Svoboda, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Experience-dependent+plasticity+of+dendritic+spines+in+the+developing+rat+barrel+cortex+in+vivo.">Experience-dependent plasticity of dendritic spines in the developing rat barrel cortex in vivo.</a> Nature. 404, 876-881 (2000).</li><li>Trachtenberg, J. T., Chen, B., Knott, G. W., Feng, G., Sanes, J. R., Welker, E., Svoboda, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-term+in+vivo+imaging+of+experience-dependent+synaptic+plasticity+in+adult+cortex.">Long-term in vivo imaging of experience-dependent synaptic plasticity in adult cortex.</a> Nature. 420, 788-794 (2002).</li><li>Portera-Cailliau, C., Weimer, R. M., DePaola, V., Caroni, P., Svoboda, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diverse+modes+of+axon+elaboration+in+the+developing+neocortex.">Diverse modes of axon elaboration in the developing neocortex.</a> PLoS Biol. 3, (2005).</li><li>Holtmaat, A., Wilbrecht, L., Knott, G. W., Welker, E., Svoboda, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Experience-dependent+and+cell-type-specific+spine+growth+in+the+neocortex.">Experience-dependent and cell-type-specific spine growth in the neocortex.</a> Nature. 441, 979-983 (2006).</li></ol>

我々はビデオでと補足の図で示したように、二光子顕微鏡の使用と組み合わせて頭蓋窓の準備は、、in vivoで新皮質の構造と機能を研究する非常に強力なツールです。テクニックは、関連する解剖学的構造と微細な外科的処置とこの準備が必要とするスキルに精通する厳しい訓練が必要です。だけ自然のままの手術は、慢性的イメージングに使用することができます。硬膜が過度またはパンクした操作される...

チタンのバーはカスタムメイドです。

a craniotomy surgery procedure for chronic brain imaging

イメージング技術は、研究の脳機能においてますます重要になっています。それはライブをそのまま脳の研究を可能にするためそれらの間で、二光子レーザー走査顕微鏡は、非常に有用な方法として浮上している。適切な準備を、このテクニックは、数分から数ヶ月に、慢性的に同一の皮質領域の観察が可能になります。このビデオでは、我々は二光子顕微鏡を用いて脳のin vivoイメージング慢性の準備を示しています。このテクニックは、最初に現在Janeliaファームでハワードヒューズ医学研究所の研究者である博士カレルスボボダ、によって開拓された。ここに示したような調製物は、カルシウム感受性色素を用いて神経活動を記録するために、（例えば、樹状突起と軸索の力学）新皮質の構造のイメージングに使用することができます、画像皮質血流の力学への、または内因性光学イメージングの研究のために。新皮質の深い画像が最適な頭蓋窓の手術で可能です。手術時の硬膜を損傷しないように細心の注意を払って使用してと一緒に、感染症を避けるために、可能な限り無菌条件下で動作し、成功と長期的なガラス張りの頭蓋のウィンドウになります。

Watch this Scientific Journal Video about A Craniotomy Surgery Procedure for Chronic Brain Imaging at JoVE.com

A Craniotomy Surgery Procedure for Chronic Brain Imaging

このビデオとプロトコルは、げっ歯類のガラスで覆われた頭蓋窓を移植する方法を示します。これらの製剤は、数ヶ月の時間スケール上の新皮質のin vivo二光子励起イメージングで慢性的に使用することができます。また、光内因性信号のイメージングなどのイメージング、他の種類、使用することができる。

慢性脳イメージングのための開頭手術の手順

Imaging techniques are becoming increasingly important in the study brain function. Among them, two-photon laser scanning microscopy has emerged as an ...

a-craniotomy-surgery-procedure-for-chronic-brain-imaging

Research

JoVE Journal

Biology

63.6K ビュー.  University of California, Los Angeles. このビデオとプロトコルは、げっ歯類のガラスで覆われた頭蓋窓を移植する方法を示します。これらの製剤は、数ヶ月の時間スケール上の新皮質のin vivo二光子励起イメージングで慢性的に使用することができます。また、光内因性信号のイメージングなどのイメージング、他の種類、使用することができる。

ビデオ: A Craniotomy Surgery Procedure for Chronic Brain Imaging

Application of Light-cured Dental Adhesive Resin for Mounting Electrodes or Microdialysis Probes in Chronic Experiments

In chronic recording experiments, self-curing dental acrylic resins have been used as a mounting base of electrodes or microdialysis-probes. Since these acrylics do not bond to the bone, screws have been used as anchors. However, in small experimental animals like finches or mouse, their craniums are very fragile and can not successfully hold the anchors. In this report, we propose a new application of light-curing dental resins for mounting base of electrodes or microdialysis probes in chronic experiments. This material allows direct bonding to the cranium. Therefore, anchor screws are not required and surgical field can be reduced considerably. Past experiences show that the bonding effect maintains more than 2 months. Conventional resin's window of time when the materials are pliable and workable is a few minutes. However, the window of working time for these dental adhesives is significantly wider and adjustable.

In this report, we propose a new application of light-curing dental resins for mounting base of electrodes or microdialysis probes in chronic experiments. This material allows direct bonding to the cranium.

慢性実験における実装電極やマイクロダイアリシスプローブ用光重合型歯科用接着性レジンの応用

In chronic recording experiments, self-curing dental acrylic resins have been used as a mounting base of electrodes or microdialysis-probes. Since these ...

生物学

Micro-drive Array for Chronic in vivo Recording: Drive Fabrication

Chronic recording of large populations of neurons is a valuable technique for studying the function of neuronal circuits in awake behaving rats. Lightweight recording devices carrying a high density array of tetrodes allow for the simultaneous monitoring of the activity of tens to hundreds of individual neurons. Here we describe a protocol for the fabrication of a micro-drive array with twenty one independently movable micro-drives. This device has been used successfully to record from hippocampal and cortical neurons in our lab. We show how to prepare a custom designed, 3-D printed plastic base that will hold the micro-drives. We demonstrate how to construct the individual micro-drives and how to assemble the complete micro-drive array. Further preparation of the drive array for surgical implantation, such as the fabrication of tetrodes, loading of tetrodes into the drive array and gold-plating, is covered in a subsequent video article.

Micro-drive Array for Chronic in vivo Recording: Drive Fabrication

In this protocol we demonstrate how to fabricate a micro-drive array for chronic electrophysiological recordings in rats.

慢性のためのマイクロドライブアレイ in vivoで録音：ドラ​​イブの作製

Chronic recording of large populations of neurons is a valuable technique for studying the function of neuronal circuits in awake behaving rats. ...

Micro-drive Array for Chronic in vivo Recording: Tetrode Assembly

The tetrode, a bundle of four electrodes, has proven to be a valuable tool for the simultaneous recording of multiple neurons in-vivo.  The differential amplitude of action potential signatures over the channels of a tetrode allows for the isolation of single-unit activity from multi-unit signals.  The ability to precisely control the stereotaxic location and depth of the tetrode is critical for studying coordinated neural activity across brain regions.  In combination with a micro-drive array, it is possible to achieve precise placement and stable control of many tetrodes over the course of days to weeks.  In this protocol, we demonstrate how to fabricate and condition tetrodes using basic tools and materials, install the tetrodes into a multi-drive tetrode array for chronic in-vivo recording in the rat, make ground wire connections to the micro-drive array, and attach a protective cone onto the micro-drive array in order to protect the tetrodes from physical contact with the environment.



Micro-drive Array for Chronic in vivo Recording: Tetrode Assembly

In this protocol we demonstrate how to fabricate and condition tetrodes for use with a micro-drive array, which was designed for chronic electrophysiological recordings in rats. In addition, we illustrate the final stages of micro-drive array construction, which includes installing ground wires and a protective cone.

慢性のためのマイクロドライブアレイ in vivoで録音：四極管アセンブリ

The tetrode, a bundle of four electrodes, has proven to be a valuable tool for the simultaneous recording of multiple neurons in-vivo.  The differential ...

Murine Renal Transplantation Procedure

Renal orthotopic transplantation in mice is a technically challenging procedure. Although the first kidney transplants in mice were performed by Russell et al over 30 years ago (1) and refined by Zhang et al years later (2), few people in the world have mastered this procedure. In our laboratory we have successfully performed 1200 orthotopic kidney transplantations with &gt; 90% survival rate. The key points for success include stringent control of reperfusion injury, bleeding and thrombosis, both during the procedure and post-transplantation, and use of 10-0 instead of 11-0 suture for anastomoses.
Post-operative care and treatment of the recipient is extremely important to transplant success and evaluation. All renal graft recipients receive antibiotics in the form of an injection of penicillin immediately post-transplant and sulfatrim in the drinking water continually. Overall animal health is evaluated daily and whole blood creatinine analyses are performed routinely with a portable I-STAT machine to assess graft function.

Renal transplantation in mice is a technically challenging procedure that requires careful post-operative care and treatment for success.

マウス腎移植の手順

Renal orthotopic transplantation in mice is a technically challenging procedure. Although the first kidney transplants in mice were performed by Russell ...

Live Imaging of the Zebrafish Embryonic Brain by Confocal Microscopy

In this video, we demonstrate the method our lab has developed to analyze the cell shape changes and rearrangements required to bend and fold the developing zebrafish brain (Gutzman et al, 2008). Such analysis affords a new understanding of the underlying cell biology required for development of the 3D structure of the vertebrate brain, and significantly increases our ability to study neural tube morphogenesis. The embryonic zebrafish brain is shaped beginning at 18 hours post fertilization (hpf) as the ventricles within the neuroepithelium inflate. By 24 hpf, the initial steps of neural tube morphogenesis are complete. Using the method described here, embryos at the one cell stage are injected with mRNA encoding membrane-targeted green fluorescent protein (memGFP). After injection and incubation, the embryo, now between 18 and 24 hpf, is mounted, inverted, in agarose and imaged by confocal microscopy. Notably, the zebrafish embryo is transparent making it an ideal system for fluorescent imaging. While our analyses have focused on the midbrain-hindbrain boundary and the hindbrain, this method could be extended for analysis of any region in the zebrafish to a depth of 
80-100 &mu;m.

In this video, we demonstrate a method by which to analyze the developing vertebrate brain in live zebrafish embryos at single cell resolution by confocal microscopy. This includes the method by which we inject the single-cell zebrafish embryo and subsequently mount and image the developing brain.

共焦点顕微鏡によるゼブラフィッシュ胚の脳のイメージングを生きる

In this video, we demonstrate the method our lab has developed to analyze the cell shape changes and rearrangements required to bend and fold the ...

Making MR Imaging Child's Play - Pediatric Neuroimaging Protocol, Guidelines and Procedure

Within the last decade there has been an increase in the use of structural and functional magnetic resonance imaging (fMRI) to investigate the neural basis of human perception, cognition and behavior 1, 2. Moreover, this non-invasive imaging method has grown into a tool for clinicians and researchers to explore typical and atypical brain development. Although advances in neuroimaging tools and techniques are apparent, (f)MRI in young pediatric populations remains relatively infrequent 2. Practical as well as technical challenges when imaging children present clinicians and research teams with a unique set of problems 3, 2. To name just a few, the child participants are challenged by a need for motivation, alertness and cooperation. Anxiety may be an additional factor to be addressed. Researchers or clinicians need to consider time constraints, movement restriction, scanner background noise and unfamiliarity with the MR scanner environment2,4-10. A progressive use of functional and structural neuroimaging in younger age groups, however, could further add to our understanding of brain development. As an example, several research groups are currently working towards early detection of developmental disorders, potentially even before children present associated behavioral characteristics e.g.11. Various strategies and techniques have been reported as a means to ensure comfort and cooperation of young children during neuroimaging sessions. Play therapy 12, behavioral approaches 13, 14,15, 16-18 and simulation 19, the use of mock scanner areas 20,21, basic relaxation 22 and a combination of these techniques 23 have all been shown to improve the participant's compliance and thus MRI data quality. Even more importantly, these strategies have proven to increase the comfort of families and children involved 12. One of the main advances of such techniques for the clinical practice is the possibility of avoiding sedation or general anesthesia (GA) as a way to manage children's compliance during MR imaging sessions 19,20. In the current video report, we present a pediatric neuroimaging protocol with guidelines and procedures that have proven to be successful to date in young children.

Making MR Imaging Child&#039;s Play - Pediatric Neuroimaging Protocol, Guidelines and Procedure

Despite an increase in the use of structural and functional magnetic resonance imaging (fMRI) in humans, the study of young pediatric populations remains a challenge. We present a hands-on, step-by-step video protocol including guidelines for clinicians and researchers intending to perform (f)MRI in young children.

小児神経イメージングプロトコル、ガイドラインと手順 - MRイメージングチャイルドプレイを作る

Within the last decade there has been an increase in the use of structural and functional magnetic resonance imaging (fMRI) to investigate the neural ...

Bioluminescence Imaging of Heme Oxygenase-1 Upregulation in the Gua Sha Procedure

Gua Sha is a traditional Chinese folk therapy that employs skin scraping to cause subcutaneous microvascular blood extravasation and bruises. The protocol for bioluminescent optical imaging of HO-1-luciferase transgenic mice reported in this manuscript provides a rapid in vivo assay of the upregulation of the heme oxygenase-1 (HO-1) gene expression in response to the Gua Sha procedure. HO-1 has long been known to provide cytoprotection against oxidative stress. The upregulation of HO-1, assessed by the bioluminescence output, is thought to represent an antioxidative response to circulating hemoglobin products released by Gua Sha. Gua Sha was administered by repeated strokes of a smooth spoon edge over lubricated skin on the back or other targeted body part of the transgenic mouse until petechiae (splinter hemorrhages) or ecchymosis (bruises) indicative of extravasation of blood from subcutaneous capillaries was observed. After Gua Sha, bioluminescence imaging sessions were carried out daily for several days to follow the dynamics of HO-1 expression in multiple internal organs.

Gua Sha, traditional Chinese therapeutic skin scraping, causes subcutaneous microvascular blood extravasation. We report a protocol of bioluminescence imaging of HO-1-luciferase transgenic mice to demonstrate that Gua Sha upregulates heme oxygenase-1 (HO-1) in multiple organs.

瓜沙手続におけるヘムオキシゲナーゼ-1アップレギュレーションの生物発光イメージング

Gua Sha is a traditional Chinese folk therapy that employs skin scraping to cause subcutaneous microvascular blood extravasation and bruises. The protocol ...

Mapping the After-effects of Theta Burst Stimulation on the Human Auditory Cortex with Functional Imaging

Auditory cortex pertains to the processing of sound, which is at the basis of speech or music-related processing1. However, despite considerable recent progress, the functional properties and lateralization of the human auditory cortex are far from being fully understood. Transcranial Magnetic Stimulation (TMS) is a non-invasive technique that can transiently or lastingly modulate cortical excitability via the application of localized magnetic field pulses, and represents a unique method of exploring plasticity and connectivity. It has only recently begun to be applied to understand auditory cortical function 2. 
An important issue in using TMS is that the physiological consequences of the stimulation are difficult to establish. Although many TMS studies make the implicit assumption that the area targeted by the coil is the area affected, this need not be the case, particularly for complex cognitive functions which depend on interactions across many brain regions 3. One solution to this problem is to combine TMS with functional Magnetic resonance imaging (fMRI). The idea here is that fMRI will provide an index of changes in brain activity associated with TMS. Thus, fMRI would give an independent means of assessing which areas are affected by TMS and how they are modulated 4. In addition, fMRI allows the assessment of functional connectivity, which represents a measure of the temporal coupling between distant regions. It can thus be useful not only to measure the net activity modulation induced by TMS in given locations, but also the degree to which the network properties are affected by TMS, via any observed changes in functional connectivity.
Different approaches exist to combine TMS and functional imaging according to the temporal order of the methods. Functional MRI can be applied before, during, after, or both before and after TMS. Recently, some studies interleaved TMS and fMRI in order to provide online mapping of the functional changes induced by TMS 5-7. However, this online combination has many technical problems, including the static artifacts resulting from the presence of the TMS coil in the scanner room, or the effects of TMS pulses on the process of MR image formation. But more importantly, the loud acoustic noise induced by TMS (increased compared with standard use because of the resonance of the scanner bore) and the increased TMS coil vibrations (caused by the strong mechanical forces due to the static magnetic field of the MR scanner) constitute a crucial problem when studying auditory processing. 
This is one reason why fMRI was carried out before and after TMS in the present study. Similar approaches have been used to target the motor cortex 8,9, premotor cortex 10, primary somatosensory cortex 11,12 and language-related areas 13, but so far no combined TMS-fMRI study has investigated the auditory cortex. The purpose of this article is to provide details concerning the protocol and considerations necessary to successfully combine these two neuroscientific tools to investigate auditory processing. 
Previously we showed that repetitive TMS (rTMS) at high and low frequencies (resp. 10 Hz and 1 Hz) applied over the auditory cortex modulated response time (RT) in a melody discrimination task 2. We also showed that RT modulation was correlated with functional connectivity in the auditory network assessed using fMRI: the higher the functional connectivity between left and right auditory cortices during task performance, the higher the facilitatory effect (i.e. decreased RT) observed with rTMS. However those findings were mainly correlational, as fMRI was performed before rTMS. Here, fMRI was carried out before and immediately after TMS to provide direct measures of the functional organization of the auditory cortex, and more specifically of the plastic reorganization of the auditory neural network occurring after the neural intervention provided by TMS. 
Combined fMRI and TMS applied over the auditory cortex should enable a better understanding of brain mechanisms of auditory processing, providing physiological information about functional effects of TMS. This knowledge could be useful for many cognitive neuroscience applications, as well as for optimizing therapeutic applications of TMS, particularly in auditory-related disorders.

Auditory processing is the basis of speech and music-related processing. Transcranial Magnetic Stimulation (TMS) has been used successfully to study cognitive, sensory and motor systems but has rarely been applied to audition. Here we investigated TMS combined with functional Magnetic Resonance Imaging to understand the functional organization of auditory cortex.

機能イメージングによるヒト聴覚皮質上のシータバースト刺激の後遺症のマッピング

Auditory cortex pertains to the processing of sound, which is at the basis of speech or music-related processing1. However, despite considerable recent ...

Isolation of Human Hepatocytes by a Two-step Collagenase Perfusion Procedure

The liver, an organ with an exceptional regeneration capacity, carries out a wide range of functions, such as detoxification, metabolism and homeostasis. As such, hepatocytes are an important model for a large variety of research questions. In particular, the use of human hepatocytes is especially important in the fields of pharmacokinetics, toxicology, liver regeneration and translational research. Thus, this method presents a modified version of a two-step collagenase perfusion procedure to isolate hepatocytes as described by Seglen 1.
Previously, hepatocytes have been isolated by mechanical methods. However, enzymatic methods have been shown to be superior as hepatocytes retain their structural integrity and function after isolation. This method presented here adapts the method designed previously for rat livers to human liver pieces and results in a large yield of hepatocytes with a viability of 77&plusmn;10%. The main difference in this procedure is the process of cannulization of the blood vessels. Further, the method described here can also be applied to livers from other species with comparable liver or blood vessel sizes.

A modified two-step collagenase perfusion procedure for isolation of human hepatocytes is described. This method can also be applied to other mammalian livers. The isolated hepatocytes are available in high yield and viability, making them a suitable model for scientific research in areas such as liver regeneration, pharmacokinetics and toxicology.

二段階コラゲナーゼ灌流手順によって、ヒト肝細胞の分離

The liver, an organ with an exceptional regeneration capacity, carries out a wide range of functions, such as detoxification, metabolism and homeostasis. ...

Investigating Protein-protein Interactions in Live Cells Using Bioluminescence Resonance Energy Transfer

Assays based on Bioluminescence Resonance Energy Transfer (BRET) provide a sensitive and reliable means to monitor protein-protein interactions in live cells. BRET is the non-radiative transfer of energy from a 'donor' luciferase enzyme to an 'acceptor' fluorescent protein. In the most common configuration of this assay, the donor is Renilla reniformis luciferase and the acceptor is Yellow Fluorescent Protein (YFP). Because the efficiency of energy transfer is strongly distance-dependent, observation of the BRET phenomenon requires that the donor and acceptor be in close proximity. To test for an interaction between two proteins of interest in cultured mammalian cells, one protein is expressed as a fusion with luciferase and the second as a fusion with YFP. An interaction between the two proteins of interest may bring the donor and acceptor sufficiently close for energy transfer to occur. Compared to other techniques for investigating protein-protein interactions, the BRET assay is sensitive, requires little hands-on time and few reagents, and is able to detect interactions which are weak, transient, or dependent on the biochemical environment found within a live cell. It is therefore an ideal approach for confirming putative interactions suggested by yeast two-hybrid or mass spectrometry proteomics studies, and in addition it is well-suited for mapping interacting regions, assessing the effect of post-translational modifications on protein-protein interactions, and evaluating the impact of mutations identified in patient DNA.

Interactions between proteins are fundamental to all cellular processes. Using Bioluminescence Resonance Energy Transfer, the interaction between a pair of proteins can be monitored in live cells and in real time. Furthermore, the effects of potentially pathogenic mutations can be assessed.

生物発光共鳴エネルギー転移を用いて生細胞中のタンパク質 - タンパク質相互作用を調査する

Assays based on Bioluminescence Resonance Energy Transfer (BRET) provide a sensitive and reliable means to monitor protein-protein interactions in live ...