资助这项研究是由林雪平大学和东约特兰省县议会。

1。去势通过背路线<ol><li>麻醉动物，例如1.5％异氟醚诱导的（4％），氧/氧化亚氮混合30％/ 70％。将动物俯卧在直肠温度计连接到一个加热板。重要的是有一层薄薄的动物和板块之间的隔离，温度计反馈系统的体温保持在37±0.5°C。申请保护眼睛凝胶和管理5毫克Rimadyl的/公斤体重生理盐水稀释皮下注射动物的脖子上缓解术后疼痛的侧面。 </li><li>剃一个4 * 4厘米（3×3厘米）对小鼠面积cephally从髂骨。彻底用例如Jodopax的光头面积覆盖的动物有一个4 * 4厘米孔径手术表之前。 </li><li>正中切口2-3厘米，并直言不讳地剖析了从底层的筋膜皮肤。中线外侧一厘米，再作切口throUGH筋膜。直言不讳地剖析横向表面尽可能根据本筋膜，直至到达腹腔。 </li><li>使用镊子，抢的，在腹腔内的卵巢周围的脂肪组织，并轻轻将其拉出。鉴别卵巢结扎子宫角和船只0.5-1厘米，这种结构近端。切卵巢和结扎脂肪组织，并投入其余组织回腹腔。另一个切口，并在对侧筋膜，重复上述步骤。 </li><li>关闭伤口，最好用单丝缝合，让从麻醉中醒来的动物。在激烈的笼（25-27℃）放置在手术后至少2小时的动物。动物应该分别被安置术后第一天。笼经常清洗/替换在恢复阶段，这是关键。手术后二十四小时内，注入动物皮下，另外5毫克稀释Rimadyl /公斤体重生理盐水。 </li></ol> 2。通过硅橡胶胶囊皮下注射17β-雌二醇<ol><li>彻底混合麻油17β-雌二醇所需金额。在大鼠和小鼠的生理浓度，我们建议180μg/ mL和18-36微克/毫升。安慰剂胶囊填充纯香油。 </li><li>切3厘米（2厘米）对小鼠的硅橡胶管的长度和5毫米（3毫米小鼠）木制涂药棒的长度为堵塞。使用镊子，挤入硅橡胶囊前注射的17β-estradiol/sesame的石油解决方案，填补了整个长度一个木塞。轻轻的开口端帽硅橡胶囊与其他木插头（插头是否应该永久地留在胶囊）。在余下的17β-雌二醇的解决方案植入前过夜孵育胶囊。 </li><li>植入胶囊可在大鼠卵巢切除术使用麻醉期间进行。为MICE，然而，卵巢切除后14天的恢复期，强烈建议胶囊通过脆弱的皮肤突起，以尽量减少风险。 </li><li> 1.1或关闭后，卵巢切除术切口手术准备后，对动物的脖子，背方面刮胡子，洗一个2 * 2厘米的面积。做一个1厘米的切口，并直言解剖皮下口袋caudolaterally充足的空间胶囊。擦拭硅橡胶囊（直到这个时间点已培养在17β-雌二醇的解决方案），并轻轻地安装它通过小切口，随后缝合封闭。这是关键，以确保胶囊株尽可能少的皮肤，特别是在靠近伤口的面积。从植入胶囊的生产，它是重要的，避免污染。 </li></ol> 3。 17β-雌二醇口服政府通过坚果，奶油花生酱<ol><li>彻底解散17β-雌二醇香油，然后与花生酱混合麻油。我们建议每天的部分包含17β-雌二醇28微克，5μL香油1克每公斤老鼠体重1.12微克17β-雌二醇，0.312μL香油和60毫克每30克鼠标花生酱花生酱。 17β-雌二醇只是没有相同的安慰剂坚果，奶油准备。 </li><li>训练动物实验前五天无激素螺母冰淇淋吃。首3天的培训中，大鼠优化应该被训练组（约0.5克的花生酱，以2-5动物），在他们家的笼子里。在第四次和第五次天的培训，动物是在单独的笼子前接受日常生活的一部分，类似的实验情况。 </li><li>在实验过程中，在单独的笼子里的动物，每饲养之际，后担任小瓷砖，其中花生酱的部分。如果训练已执行以上，O你的经验是，超过95％的大鼠和小鼠接受坚果冰淇淋，一旦完全习惯于，大多数动物在几秒钟内消耗。 </li></ol> 4。代表结果生理血清17β-雌二醇的浓度，而目前的方法旨在重现，约5-140皮克/毫升大鼠和小鼠4,6 5-35皮克/毫升。皮下的硅胶胶囊，根据上述指示，当300克只雌性SD大鼠，产生缓慢下降17β-雌二醇的血药浓度约40皮克/毫升一天2至10皮克/毫升35天。然而，立即给药后，约1000皮克/毫升的血药浓度高峰，可以预期，在8小时内下降到生理范围内（ 图1A）4,5。 36的微克17β-estradiol/mL，管理时至25克雌性C57BL / 6小鼠以上所述的小鼠，硅橡胶胶囊，呈现血清浓entrations缓慢下降约90皮克/毫升每天2至25皮克/毫升35天。这可能是由一个类似的高峰之前，被发现在大鼠，虽然这并没有被查处（ 图1B）6。 口服剂量的花生酱上面介绍的方法，当300克只雌性SD大鼠，呈现17β-雌二醇10-70皮克/毫升（ 图2A）4之间的每日波动的生理水平。对于使用上述剂量的小鼠，每日水平波动之间的约20和120皮克/毫升（ 图2B）6。 <img alt="图1" src="/files/ftp_upload/4013/4013fig1.jpg" /> <st<html>荣&gt;图1。程式化的图形呈现17β-雌二醇浓度4,5（A，N = 10个时间点多达24个小时，在以后的时间点N = 15）在大鼠和小鼠（B，N = 10） 6胶囊植入后。硅橡胶的胶囊生产短的初始峰值的血药浓度达1000皮克/毫升，但在几个小时内下降到100皮克/毫升以下的水平。在未来4-5周，胶囊产量稳定，虽然持续下降，血药浓度。阴影部分对应的生理17β-雌二醇完整的雌性大鼠和小鼠的血清浓度。即使没有视觉图表显示，在17β-雌二醇浓度间的个体差异，使变异系数从27到183％和大鼠从28至115％的硅橡胶囊方法小鼠（非常低，平均浓度往往产生变异系数高）。 iles/ftp_upload/4013/4013fig2.jpg“/&gt; 图2展示17β-雌二醇浓度在大鼠的程式化的图（A，N = 15）。4和小鼠（B，N = 10）6，花生酱每日激素管理的后果。每天口服花生酱的方法呈现高峰，但不是通过远高皮下或静脉注射的方法制作。这模仿人类的​​激素替代疗法的药物动力学，口服治疗可以进行，只要需要，即使上面的例子只提出了一个最初的96小时。阴影部分对应的生理17β-雌二醇完整的雌性大鼠和小鼠的血清浓度。即使不直观图表显示，在17β-雌二醇浓度间的个体差异，使在小鼠花生酱的方法从大鼠的40％至132％，从13到219％，变异系数（平均浓度非常低往往产生高变异系数）。

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作者没有透露。

上面介绍的管理模式，在早先的研究证明优于商业缓释微丸生产生理血清浓度17β-雌二醇注射。而缓释微丸生产非常高，长期的血药浓度峰值，注射导致极端情况下，每天的激素的波动，需要反复的动物4-6压力处理。选择适当的管理模式的重要性，强调了最近的研究表明，虽然低剂量的17β-雌二醇的管理方案（如在目前的文章中提出的技术）防止脑缺血8-10，17β-雌二醇浓度由生产?...

<table border="1"><tbody><tr><td> 试剂名称 </td><td> 公司 </td><td> 目录编号 </td><td> 评论 </td></tr><tr><td> rimadyl，50毫克/毫升</td><td>辉瑞公司</td><td> 462986 </td><td>卵巢切除后的术后镇痛。 </td></tr><tr><td> lubrithal眼凝胶</td><td>狮子座实验室</td><td></td><td>在手术过程中注意保护眼睛。 </td></tr><tr><td> jodopax兽医</td><td> pharmaxim </td><td></td><td>手术消毒。 </td></tr><tr><td> vicryl 4-0缝合</td><td>强生公司</td><td></td><td>伤口缝合和卵巢结扎。 </td></tr><tr><td> 17β-雌二醇，&gt; 98％ </td><td>西格玛</td><td> E8875-1G </td><td>在硅橡胶粒和花生酱麻油。 </td></tr><tr><td>香油</td><td>西格玛</td><td> S3547-1L </td><td>解散17β-雌二醇。 </td></tr><tr><td>硅橡胶管，内径/外径：1.575/3.175毫米</td><td>道康宁</td><td> 508-008 </td><td>硅胶胶囊的生产。 </td></tr><tr><td>木制涂药棒</td><td> selefatrade </td><td> 12-0783 </td><td>封顶的硅胶胶囊。 </td></tr><tr><td>花生酱</td><td>费雷罗</td><td></td><td>口服17β-雌二醇的培养基。 </td></tr></tbody></table>

ovariectomy and 17&beta;-estradiol replacement in rats and mice: a visual demonstration

雌激素是女性性激素的影响格外广泛的家庭。当大鼠和小鼠使用雌激素的研究，它们通常以消融迅速骑车激素生产，更换外源性17β-雌二醇去势。有，然而，缺乏有关激素应如何管理，以获取生理血清浓度的共识。这是至关重要的，因为17β-雌二醇的水平/管理方法深刻地影响实验结果1-3。我们有一系列特点17β-雌二醇管理的不同模式的研究，发现皮下硅胶胶囊和每口螺母，奶油花生酱均优于市售缓释微丸（创新研究由美国公司生产）在生产生理血清浓度的17β-雌二醇4-6每日注射。当中高级坚果冰淇淋的方法，以前一直使用丁丙诺啡管理7，antages是，当雌激素的管理使用，它类似于口服激素替代疗法和非侵入性。皮下硅胶胶囊是方便和生产最稳定的血药浓度。这部影片的文章包含了卵巢切除术的一步一步的示范和17β-雌二醇激素替代硅橡胶胶囊和口服花生酱在大鼠和小鼠，其次是行政程序的重要方面进行讨论。

Watch this Scientific Journal Video about Ovariectomy and 17Î²-estradiol Replacement in Rats and Mice: A Visual Demonstration at JoVE.com

Ovariectomy and 17&amp;#946;-estradiol Replacement in Rats and Mice: A Visual Demonstration

卵巢切除术和硅胶胶囊和口服花生酱，随后更换17β-雌二醇表明，在大鼠和小鼠。

在大鼠和小鼠的卵巢切除和17β-雌二醇更换：一个可视化示范

Estrogens are a family of female sexual hormones with an exceptionally wide spectrum of effects. When rats and mice are used in estrogen research they are ...

ovariectomy-17-estradiol-replacement-rats-mice-visual

Research

JoVE Journal

Biology

Ovariectomy and 17&beta;-estradiol Replacement in Rats and Mice: A Visual Demonstration

56.7K 次观看.  Linköping University. 卵巢切除术和硅胶胶囊和口服花生酱，随后更换17β-雌二醇表明，在大鼠和小鼠。

视频: Ovariectomy and 17&#946;-estradiol Replacement in Rats and Mice: A Visual Demonstration

A Magnetic Tether System to Investigate Visual and Olfactory Mediated Flight Control in Drosophila

It has been clear for many years that insects use visual cues to stabilize their heading in a wind stream. Many animals track odors carried in the wind. As such, visual stabilization of upwind tracking directly aids in odor tracking. But do olfactory signals directly influence visual tracking behavior independently from wind cues? Also, the recent deluge of research on the neurophysiology and neurobehavioral genetics of olfaction in Drosophila has motivated ever more technically sophisticated and quantitative behavioral assays. Here, we modified a magnetic tether system originally devised for vision experiments by equipping the arena with narrow laminar flow odor plumes. A fly is glued to a small steel pin and suspended in a magnetic field that enables it to yaw freely. Small diameter food odor plumes are directed downward over the fly s head, eliciting stable tracking by a hungry fly. Here we focus on the critical mechanics of tethering, aligning the magnets, devising the odor plume, and confirming stable odor tracking.

Here we describe how to tether a fly in an olfactory magnetic-tether (OMT) apparatus. We describe how to align the rare-earth magnets and odor ports, and how to set mass flow rates for both the stimulus delivery and vacuum suction to achieve optimal odor tracking.

一个磁性的系绳系统，以调查在果蝇的视觉和嗅觉介导的飞行控制

It has been clear for many years that insects use visual cues to stabilize their heading in a wind stream. Many animals track odors carried in the wind. ...

科研

生物学

Demonstration of Cutaneous Allodynia in Association with Chronic Pelvic Pain

Pelvic pain is a common condition that is associated with dysmenorrhea and endometriosis. In some women the severe episodes of cyclic pain change and the resultant pain becomes continuous and this condition becomes known as Chronic Pelvic Pain. This state can be present even after the appropriate medical or surgical therapy has been instituted. It can be associated with pain and tenderness in the muscles of the abdomen wall and intra-pelvic muscles leading to severe dyspareunia. Additional symptoms of irritable bowel and interstitial cystitis are common. A common sign of the development of this state is the emergence of cutaneous allodynia which emerges from the so-called viscero-somatic reflex. A simple bedside test for the presence of cutaneous allodynia is presented that does not require excessive time or special equipment. This test builds on previous work associated with changes in sensation related to gall bladder function and the viscera-somatic reflex(1;2).
The test is undertaken with the subject s permission after an explanation of how the test will be performed. Allodynia refers to a condition in which a stimulus that is not normally painful is interpreted by the subject as painful. In this instance the light touch associated with a cotton-tipped applicator would not be expected to be painful. A positive test is however noted by the woman as suddenly painful or suddenly sharp. The patterns of this sensation are usually in a discrete pattern of a dermatome of the nerves that innervate the pelvis.
The underlying pathology is now interpreted as evidence of neuroplasticity as a consequence of severe and repeating pain with changes in the functions of the dorsal horns of the spinal cord that results in altered function of visceral tissues and resultant somatic symptoms(3). 
The importance of recognizing the condition lies in an awareness that this process may present coincidentally with the initiating condition or after it has been treated. It also permits the clinician to evaluate the situation from the perspective that alternative explanations for the pain may be present that may not require additional surgery.

Demonstration of Cutaneous Allodynia in Association with Chronic Pelvic Pain

A demonstration of the bedside test for cutaneous allodynia and its clinical implications.

示范皮肤触感在慢性盆腔疼痛协会

Pelvic pain is a common condition that is associated with dysmenorrhea and endometriosis.  In some women the severe episodes of cyclic pain change and the ...

Single-cell Suction Recordings from Mouse Cone Photoreceptors

Rod and cone photoreceptors in the retina are responsible for light detection. In darkness, cyclic nucleotide-gated (CNG) channels in the outer segment are open and allow cations to flow steadily inwards across the membrane, depolarizing the cell. Light exposure triggers the closure of the CNG channels, blocks the inward cation current flow, and thus results in cell hyperpolarization. Based on the polarity of photoreceptors, a suction recording method was developed in 1970s that, unlike the classic patch-clamp technique, does not require penetrating the plasma membrane 1. Drawing the outer segment into a tightly-fitting glass pipette filled with extracellular solution allows recording the current changes in individual cells upon test-flash exposure. However, this well-established "outer-segment-in (OS-in)" suction recording is not suitable for mouse cone recordings, because of the low percentage of cones in the mouse retina (3%) and the difficulties in identifying the cone outer segments. Recently, an inner-segment-in (IS-in) recording configuration was developed to draw the inner segment/nuclear region of the photoreceptor into the recording pipette 2,3. In this video, we will show how to record from individual mouse cone photoresponses using single-cell suction electrode.

We will show how to record flash responses from single mouse cones using a suction electrode.

单从小鼠锥感光细胞吸录音

Rod and cone photoreceptors in the retina are responsible for light detection. In darkness, cyclic nucleotide-gated (CNG) channels in the outer segment ...

A Practical Approach to Genetic Inducible Fate Mapping: A Visual Guide to Mark and Track Cells In Vivo

Fate maps are generated by marking and tracking cells in vivo to determine how progenitors contribute to specific structures and cell types in developing and adult tissue. An advance in this concept is Genetic Inducible Fate Mapping (GIFM), linking gene expression, cell fate, and cell behaviors in vivo, to create fate maps based on genetic lineage.
GIFM exploits X-CreER lines where X is a gene or set of gene regulatory elements that confers spatial expression of a modified bacteriophage protein, Cre recombinase (CreERT). CreERT contains a modified estrogen receptor ligand binding domain which renders CreERT sequestered in the cytoplasm in the absence of the drug tamoxifen. The binding of tamoxifen releases CreERT, which translocates to the nucleus and mediates recombination between DNA sequences flanked by loxP sites. In GIFM, recombination typically occurs between a loxP flanked Stop cassette preceding a reporter gene such as GFP.
Mice are bred to contain either a region- or cell type-specific CreER and a conditional reporter allele. Untreated mice will not have marking because the Stop cassette in the reporter prevents further transcription of the reporter gene. We administer tamoxifen by oral gavage to timed-pregnant females, which provides temporal control of CreERT release and subsequent translocation to the nucleus removing the Stop cassette from the reporter. Following recombination, the reporter allele is constitutively and heritably expressed. This series of events marks cells such that their genetic history is indelibly recorded. The recombined reporter thus serves as a high fidelity genetic lineage tracer that, once on, is uncoupled from the gene expression initially used to drive CreERT.
We apply GIFM in mouse to study normal development and ascertain the contribution of genetic lineages to adult cell types and tissues. We also use GIFM to follow cells on mutant genetic backgrounds to better understand complex phenotypes that mimic salient features of human genetic disorders.
This video article guides researchers through experimental methods to successfully apply GIFM. We demonstrate the method using our well characterized Wnt1-CreERT;mGFP mice by administering tamoxifen at embryonic day (E)8.5 via oral gavage followed by dissection at E12.5 and analysis by epifluorescence stereomicroscopy. We also demonstrate how to micro-dissect fate mapped domains for explant preparation or FACS analysis and dissect adult fate-mapped brains for whole mount fluorescent imaging. Collectively, these procedures allow researchers to address critical questions in developmental biology and disease models.

A Practical Approach to Genetic Inducible Fate Mapping: A Visual Guide to Mark and Track Cells In Vivo

Genetic Inducible Fate Mapping (GIFM) marks and tracks cells with fine spatial and temporal control in vivo and elucidates how cells from a specific genetic lineage contribute to developing and adult tissues. Demonstrated here are the techniques required to fate map E12.5 mouse embryos for epifluorescent and explant analysis.

一个实用的方法遗传诱导的命运映射：一个视觉引导标记和跟踪细胞在体内</em

Fate maps are generated by marking and tracking cells in vivo to determine how progenitors contribute to specific structures and cell types in developing ...

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 ...

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 ...

Design and Analysis of Temperature Preference Behavior and its Circadian Rhythm in Drosophila

The circadian clock regulates many aspects of life, including sleep, locomotor activity, and body temperature (BTR) rhythms1,2. We recently identified a novel Drosophila circadian output, called the temperature preference rhythm (TPR), in which the preferred temperature in flies rises during the day and falls during the night 3. Surprisingly, the TPR and locomotor activity are controlled through distinct circadian neurons3. Drosophila locomotor activity is a well&#160;known circadian behavioral output and has provided strong contributions to the discovery of many conserved mammalian circadian clock genes and mechanisms4. Therefore, understanding TPR will lead to the identification of hitherto unknown molecular and cellular circadian mechanisms. Here, we describe how to perform and analyze the TPR assay. This technique not only allows for dissecting the molecular and neural mechanisms of TPR, but also provides new insights into the fundamental mechanisms of the brain functions that integrate different environmental signals and regulate animal behaviors. Furthermore, our recently published data suggest that the fly TPR shares features with the mammalian BTR3. Drosophila are ectotherms, in which the body temperature is typically behaviorally regulated. Therefore, TPR is a strategy used to generate a rhythmic body temperature in these flies5-8. We believe that further exploration of Drosophila TPR will facilitate the characterization of the mechanisms underlying body temperature control in animals.

Design and Analysis of Temperature Preference Behavior and its Circadian Rhythm in Drosophila

We recently identified a novel Drosophila circadian output, temperature preference rhythm (TPR), in which the preferred temperature in flies rises during the day and falls during the night. TPR is regulated independently from another circadian output, locomotor activity. Here&#160;we describe the design and analysis of TPR in Drosophila.

温度偏好行为及其在德罗索菲拉的昼夜节律的设计与分析

The circadian clock regulates many aspects of life, including sleep, locomotor activity, and body temperature (BTR) rhythms1,2. We recently identified a ...

Physiology Lab Demonstration: Glomerular Filtration Rate in a Rat

Measurements of glomerular filtration rate (GFR), and the fractional excretion of sodium (Na) and potassium (K) are critical in assessing renal function in health and disease. GFR is measured as the steady state renal clearance of inulin which is filtered at the glomerulus, but not secreted or reabsorbed along the nephron. The fractional excretion of Na and K can be determined from the concentration of Na and K in plasma and urine. The renal clearance of inulin can be demonstrated in an anesthetized animal which has catheters in the femoral artery, femoral vein and bladder. The equipment and supplies used for this procedure are those commonly available in a research core facility, and thus makes this procedure a practical means for measuring renal function. The purpose of this video is to demonstrate the procedures required to perform a lab demonstration in which renal function is assessed before and after a diuretic drug. The presented technique can be utilized to assess renal function in rat models of renal disease.

The purpose of this protocol is to demonstrate the principles and techniques for measuring and calculating glomerular filtration rate, urine flow rate, and excretion of sodium and potassium in a rat. This demonstration can be used to provide students with an overall conceptual understanding of how to measure renal function.

生理学实验室演示：肾小球滤过率在一个老鼠

Measurements of glomerular filtration rate (GFR), and the fractional excretion of sodium (Na) and potassium (K) are critical in assessing renal function ...

Validation of a Mouse Model to Disrupt LINC Complexes in a Cell-specific Manner

Nuclear migration and anchorage within developing and adult tissues relies heavily upon large macromolecular protein assemblies called LInkers of the Nucleoskeleton and Cytoskeleton (LINC complexes). These protein scaffolds span the nuclear envelope and connect the interior of the nucleus to components of the surrounding cytoplasmic cytoskeleton. LINC complexes consist of two evolutionary-conserved protein families, Sun proteins and Nesprins that harbor C-terminal molecular signature motifs called the SUN and KASH domains, respectively. Sun proteins are transmembrane proteins of the inner nuclear membrane whose N-terminal nucleoplasmic domain interacts with the nuclear lamina while their C-terminal SUN domains protrudes into the perinuclear space and interacts with the KASH domain of Nesprins. Canonical Nesprin isoforms have a variable sized N-terminus that projects into the cytoplasm and interacts with components of the cytoskeleton. This protocol describes the validation of a dominant-negative transgenic mouse strategy that disrupts endogenous SUN/KASH interactions in a cell-type specific manner. Our approach is based on the Cre/Lox system that bypasses many drawbacks such as perinatal lethality and cell nonautonomous phenotypes that are associated with germline models of LINC complex inactivation. For this reason, this model provides a useful tool to understand the role of LINC complexes during development and homeostasis in a wide array of tissues.

Nuclear envelope proteins play a central role in many basic biological processes and have been implicated in a variety of human diseases. This protocol describes a new Cre/Lox-based mouse model that allows for the spatiotemporal control of LINC complexes disruption.

小鼠模型的验证扰乱LINC配合物在细胞特异性的方式

Nuclear migration and anchorage within developing and adult tissues relies heavily upon large macromolecular protein assemblies called LInkers of the ...

In Vivo Detection and Analysis of Rb Protein SUMOylation in Human Cells

The post-translational modifications of proteins are critical for the proper regulation of intracellular signal transduction. Among these modifications, small ubiquitin-related modifier (SUMO) is a ubiquitin-like protein that is covalently attached to the lysine residues of a variety of target proteins to regulate cellular processes, such as gene transcription, DNA repair, protein interaction and degradation, subcellular transport, and signal transduction. The most common approach to detecting protein SUMOylation is based on the expression and purification of recombinant tagged proteins in bacteria, allowing for an in vitro biochemical reaction which is simple and suitable for addressing mechanistic questions. However, due to the complexity of the process of SUMOylation in vivo, it is more challenging to detect and analyze protein SUMOylation in cells, especially when under endogenous conditions. A recent study by the authors of this paper revealed that endogenous retinoblastoma (Rb) protein, a tumor suppressor that is vital to the negative regulation of the cell cycle progression, is specifically SUMOylated at the early G1 phase. This paper describes a protocol for the detection and analysis of Rb SUMOylation under both endogenous and exogenous conditions in human cells. This protocol is appropriate for the phenotypical and functional investigation of the SUMO-modification of Rb, as well as many other SUMO-targeted proteins, in human cells.

In Vivo Detection and Analysis of Rb Protein SUMOylation in Human Cells

Small ubiquitin-related modifier (SUMO) family proteins are conjugated to the lysine residues of target proteins to regulate various cellular processes. This paper describes a protocol for the detection of retinoblastoma (Rb) protein SUMOylation under endogenous and exogenous conditions in human cells.

在体内人细胞 Rb 蛋白 sumo 的检测与分析

The post-translational modifications of proteins are critical for the proper regulation of intracellular signal transduction. Among these modifications, ...