此协议是在简多德，托马斯Jessell和安德鲁拉姆斯登的实验室开发的。许多人，包括康拉德巴斯勒，安Calof，托马斯埃德伦德，菲尔汉密尔顿，Domna Karagogeos，沙龙鲁伊斯我Altaba和俊山田决定如何脊髓胶原蛋白的文化植。 Marysia Placzek和马克特西尔儿率先使用在体外植体作为识别轴突导向分子的手段的轴突的生长技术。巴特勒实验室的工作是从3月从美国国立卫生研究院/ NINDS优生优育基金会和R01 NS063999的赠款支持。

第1部分：使用悬滴转染COS细胞聚合制备<ol><li>种子COS - 7（COS）在35mm培养皿中的细胞。当他们到达80％汇合，到使用Lipofectamine2000根据制造商S协议细胞的表达质粒转染1μg。 </li><li>为了准备吊滴，吸转染介质和细胞的转染COS1毫升的1X的磷酸盐缓冲液（PBS）冲洗。治疗与无细胞酶的解离15分钟的中等0.5毫升的细胞。添加OPTI - MEM + 1青霉素/链霉素/谷氨酰胺（P / S / G）+ 10％小牛血清（FBS），停止反应的解决方案1毫升。 </li><li>磨碎的细胞培养皿的表面删除，并转移到一个15毫升锥形管。旋转2分钟沉淀细胞在2000K。删除OPTI - MEM + 1X P / S / G + 10％FBS的100μL上清，重悬沉淀。 </li><li>点上一个35mm培养皿的盖子内的多个20μL滴，反转盘底部顶部的盖子和地点，并在37 ° C培养箱离开几个小时，待细胞总。 </li></ol>第2部分：背侧脊髓外植体的制备<ol><li>从母亲的子宫解剖E11大鼠胚胎，并保持在L15的介质上的冰，直到需要。 </li><li>有了一个削尖的钨针，请从每个胚胎的躯干4-6段部分，从下方前肢芽的区域。用塑料吸管，收集在一个4以及NUNC菜的组织块，置于冰上。 </li><li>当所有的胚胎段已被解剖，用塑料吸管转移到一个解决方案，为1ml L15 + 1mg的dispase在NUNC盘的第二件。在室温下孵育时间不超过5分钟。不要过度的孵化与dispase组织块。 </li><li>在孵化期间，加入热灭活正常山羊血清（HIGS）0.5毫升L1510毫升到培养皿中，漩涡混合。转移到第三NUNC菜的这一解决方案1毫升，并转移到该解决方案的组织块，孵化结束时。置于冰上。注意：如果允许一个小时“休息”冰的组织块，随后的剥离会比较容易。 </li></ol>第3部分：底漆胶原蛋白<ol><li>加入40μl10倍最小的必不可少的中等至360μl胶原蛋白，混合迅速，彻底地轻弹或简要涡旋管。在picofuge迅速降速。该解决方案将是黄色的。，尽可能保持在冰上 。 </li><li>添加足够的0.8M的碳酸氢钠（ 碳酸氢钠3）打开略有橙色的胶原蛋白溶液（见下面的说明） 。再次，混合迅速，彻底地弹和自旋向下picofuge简要。如果解决方案仍然是粉红色混合后，你有太多碳酸氢钠 3，将需要再次开始的胶原蛋白的新鲜管。 </li><li>此时，胶原溶液催芽。它仍将在冰上的液体，但巩固带来至室温时，将在5分钟左右（车削粉红色）。 </li><li>在每一个4孔NUNC菜以及胶原蛋白的现货20μL。使用吸管的笔尖，摊开的胶原蛋白，以形成一个小型的“垫”。让我们在室温胶原。 </li></ol>注：3碳酸氢钠的确切数额将需要每批胶原蛋白的滴定。起点低（11μl），然后添加0.5 -1μl进逐步直到解决方案是一个橙色轻微阴影。 “正 ​​确”的数额通常会变成胶原蛋白粉红色的最小量的碳酸氢钠 3 1ul 。碳酸氢钠3量并不线性比例上升或下降。 第4部分：夹层背侧脊髓外植体和他们的定位与COS在胶原蛋白基质细胞聚集<ol><li>使用新鲜削尖的钨针和一双细镊子（＃5或＃55），取出脊髓周围的中胚层。 </li><li>平行切割的底板与钨针，小心取出脊髓腹侧第五。 </li><li>对开背侧脊髓外植体，确保边缘尽可能干净切。重新修剪的边缘，如果有必要。每个背侧脊髓外植体转移到单独的胶原蛋白，使用移液器装上拉至大约用玻璃毛细管管口垫。 0.5毫米直径。 </li><li>切转染COS细胞约背侧脊髓外植体的外侧边缘的宽度相同的平方的总和。 </li><li>这些广场之一转移到背侧脊髓外植体的胶原蛋白垫，使用口吸管。 </li><li>用口吸管，吸过任何多余的液体，用口吸管。不要过度吸。 </li><li>应用到催芽胶原4μl，垫，使用钨针不直接接触组织的胶原蛋白，在植。通过移动在植体周围的胶原的钨针，东方植，使COS细胞总量的背侧脊髓外植体外侧缘旁。 </li><li>胶原蛋白后，重复的胶原蛋白4μl的应用，以确保外植体完全包裹在胶原。 </li><li>在组织培养罩中，添加OPTI - MEM + 1xP/S/G和30-40小时的文化0.5毫升。 </li><li>修复植0.5毫升4％多聚甲醛在1xPBS 45分钟，冰板。 </li><li>洗两次的1xPBS封闭液+ 0.1％，1％TRITON - X100 HIGS（PBTN）0.5毫升。座在4 ° C的至少几个小时，孵育过夜，是可取的，然后通过免疫组化的过程中。 </li></ol>第5部分：通过免疫组连合轴突的可视化<ol><li> PBTN阻断后，削减的植产钳和一个48孔盘的4孔板。 </li><li>新增的主要抗体200μL，每孔，留在4℃过夜° C。为了形象化连合轴突，使用鼠抗TAG1抗体1:6的解决方案。要确定转染是否成功，还包括对被测试的信号分子，或相关的抗原表位的信号分子已被抗原表位标记的抗体。 </li><li>每个样品洗5 × 1小时PBTN在4 ° C。 </li><li>新增二级抗体200μL，每孔，留在4℃过夜° C。如果反TAG1抗体作为第一抗体，羊抗小鼠IgM二抗耦合FITC或Cy3标记。 如果加上使用光敏二级抗体FITC或Cy3标记，保持箔覆盖从这个角度上的48孔盘。 </li><li>洗净每个样品5 × 1小时PBTN 4 ° C。 </li><li>转移的外植体，以3以及抑郁症的幻灯片，删除多余的液体，Vectashield中型和盖玻片的安装。在轻保险箱商店，4 ° C或-20 ° C </li></ol>第6部分：外植体的代表图片在一个成功的实验，外植体和COS的总将保持彼此相邻，而不是疏远胶原套。会有最小的轴突的繁茂;显着的轴突的繁茂表示，背侧脊髓外植体培养时间过长。将有从外植体生长无滤过泡;出泡发生，如果组织直接接触带有培养基。 使用共聚焦显微镜的信号分子的能力，重新调整轴突评估。通过测量平均角度调整最接近的COS细胞总3的轴突，轴突转岗程度可以量化。如图1所示，COS细胞中表达myc基因标签BMP7（蓝色）重新调整TAG1连合轴突+（绿色），类似的方式，顶板的外植体排斥连合轴突（图BMP7源（图1B ） 1A）。 COS细胞中表达的控制向量3,4连合轴突的方向没有影响。这些植也被标记抗体对转录因子Isl1 / 2（红色），其中装饰运动神经元和背interneurons。这一结果首次显示了BMP家族介导成员顶板chemorepellent 3的活性。 <img src="/files/ftp_upload/1853/1853fig1tmb.jpg" border="0" /> 图1：请<a href="https://www.jove.com/files/ftp_upload/1853/1853fig1.jpg">点击这里</a>看到图1的放大版本。

<ol>
	<li>Altman, J., Bayer, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The development of the rat spinal cord. , (1984).</li><li>Placzek, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue+recombinations+in+collagen+gels.">Tissue recombinations in collagen gels.</a> Methods in molecular biology. , 325-335 (2008).</li><li>Augsburger, A., Schuchardt, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=BMPs+as+mediators+of+roof+plate+repulsion+of+commissural+neurons.">BMPs as mediators of roof plate repulsion of commissural neurons.</a> Neuron. 24, 127-141 (1999).</li><li>Butler, S. J., Dodd, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+role+for+BMP+heterodimers+in+roof+plate-mediated+repulsion+of+commissural+axons.">A role for BMP heterodimers in roof plate-mediated repulsion of commissural axons.</a> Neuron. 38, 389-401 (2003).</li><li>Kennedy, T. E., Serafini, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Netrins+are+diffusible+chemotropic+factors+for+commissural+axons+in+the+embryonic+spinal+cord.">Netrins are diffusible chemotropic factors for commissural axons in the embryonic spinal cord.</a> Cell. 78, 425-435 (1994).</li><li>Charron, F., Stein, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+morphogen+sonic+hedgehog+is+an+axonal+chemoattractant+that+collaborates+with+netrin-1+in+midline+axon+guidance.">The morphogen sonic hedgehog is an axonal chemoattractant that collaborates with netrin-1 in midline axon guidance.</a> Cell. 113, 11-23 (2003).</li><li>Lumsden, A. G., Davies, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemotropic+effect+of+specific+target+epithelium+in+the+developing+mammalian+nervous+system.">Chemotropic effect of specific target epithelium in the developing mammalian nervous system.</a> Nature. 323, 538-539 (1986).</li><li>Dodd, J., Morton, S. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spatial+regulation+of+axonal+glycoprotein+expression+on+subsets+of+embryonic+spinal+neurons.">Spatial regulation of axonal glycoprotein expression on subsets of embryonic spinal neurons.</a> Neuron. 1, 105-116 (1988).</li><li>Yamauchi, K., Phan, K. D., Butler, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=BMP+type+I+receptor+complexes+have+distinct+activities+mediating+cell+fate+and+axon+guidance+decisions.">BMP type I receptor complexes have distinct activities mediating cell fate and axon guidance decisions.</a> Development. 135, 1119-1128 (2008).</li></ol>

确定在执行这个实验成功的关键因素，首先，组织不应与太长时间的一个时期dispase治疗，这种治疗会导致组织变得很粘和减少可行性。二，胶原蛋白，必须完全底漆和保持尽可能在冰上。这将成为越来越难以处理，如果它开始设置，即变成粉红色。三，钨针必须始终保持非常尖锐的。

assaying the ability of diffusible signaling molecules to reorient embryonic spinal commissural axons

在脊椎动物的脊髓后连合轴突一直是非常宝贵的模型系统，在其中找出轴突导向信号。在这里，我们描述了一个在体外实验中，“转岗检测”，已广泛用于研究方向 2连合轴突的外在和内在信号的影响。此法简多德，托马斯Jessell和安德鲁拉姆斯登的实验室中的许多人（详情请参阅确认），这个实验版本用于演示重新定位的关键轴突导向分子的活动，包括BMP在chemorepellent顶板3,4和Netrin1 5和刺猬（SHH）在脊髓中的底板 6 chemoattractive活动。 外植体，包括2-3段脊髓背三分之二是从胚胎一天（五）11大鼠解剖，并在三维胶原凝胶 7中培养。 E11背脊髓植包含新出生的连合神经元，他们的糖蛋白，TAG1 8轴突的表达，可以通过确定的。在30-40小时在培养过程中，连合轴突轨迹是在同一个时间过程相似，在体内看到这些背植详。这轴突的轨迹，可以放置或者测试组织或一个背外植体的外侧边缘接触的候选信号分子表达的COS细胞总的挑战。连合轴突延长追加组织附近的内源性顶板和信号从横向组织异位的影响下成长。在这种情况下，重新调整的程度，连合轴突可以量化的。使用这个实验中，它可能既要研究的一个特定的信号重新调整连合轴突，以及为这个信号直接连合的轨迹9的必要性3,4自给自足。

Watch this Scientific Journal Video about Assaying the Ability of Diffusible Signaling Molecules to Reorient Embryonic Spinal Commissural Axons at JoVE.com

Assaying the Ability of Diffusible Signaling Molecules to Reorient Embryonic Spinal Commissural Axons

这种检测评估的一种信号分子的能力，这里骨形态发生蛋白7（BMP7），调整连合轴突。一个胚胎脊髓背侧的外植体是培养毗邻COS细胞分泌候选人生长因子的总和。重新调整连合轴突生长外植体内免疫组织化学显示。

含量测定扩散的信号分子能够重新调整胚胎脊髓连合轴突

Dorsal commissural axons in the vertebrate spinal cord1 have been an invaluable model system in which to identify axon guidance signals.  Here, we ...

assaying-ability-diffusible-signaling-molecules-to-reorient-embryonic

Research

JoVE Journal

Biology

8.8K 次观看.  University of Southern California. 这种检测评估的一种信号分子的能力，这里骨形态发生蛋白7（BMP7），调整连合轴突。一个胚胎脊髓背侧的外植体是培养毗邻COS细胞分泌候选人生长因子的总和。重新调整连合轴突生长外植体内免疫组织化学显示。

视频: Assaying the Ability of Diffusible Signaling Molecules to Reorient Embryonic Spinal Commissural Axons

Fabrication of a Microfluidic Device for the Compartmentalization of Neuron Soma and Axons

In this video, we demonstrate the technique of soft lithography with polydimethyl siloxane (PDMS) which we use to fabricate a microfluidic device for culturing neurons. Previously, a silicon wafer was patterned with the design for the neuron microfluidic device using SU-8 and photolithography to create a master mold, or what we simply refer to as a "master". Next, we pour the silicon polymer PDMS on top of the master which is then cured by heating the PDMS to 80&deg;C for 1 hour. The PDMS forms a negative mold of the device. The PDMS is then carefully cut and lifted away from the master. Holes are punched where the reservoirs will be and the excess PDMS trimmed away from the device. Nitrogen is used to blow away any excess debris from the device. At this point the devices are now ready for use and can either bonded to corning No. 1 cover glass with a plasma sterilizer/cleaner or can be reversibly bound to the cover glass by simply placing the device on top of the cover glass. The reversible bonding of the device to glass is covered in a separate video and requires first that the device be sterilized either with 70% ethanol or by autoclaving. Plasma treating sterilizes the devices so no further treatment is necessary. It is, however, important, when plasma-treating the devices, to add liquid to the devices within 10 minutes of the plasma treatment while the surfaces are still hydrophilic. Waiting longer than 10 minutes to add liquid to the device makes it difficult for the liquid to enter the device. The neuron devices are typically plasma-bound to cover glass and 0.5 mg/ml poly-L-lysine (PLL) in pH 8.5 borate buffer is immediately added to the device. After a minimum of 3 hours incubating with PLL, the devices are washed with dH2O water a minimum of 3 times with at least 15 minutes between each wash. Next, the water is removed and fresh media is added to the device. At this point the device is ready for use. It is important to remember at this point to never remove all the media from the device. Always leave media in the main channel.

In this video we demonstrate the technique of soft lithography with polydimethyl siloxane (PDMS) which we use to farbricate a microfluidic device for culturing neurons.

条块索玛的神经元和轴突的一个微流体设备制造

In this video, we demonstrate the technique of soft lithography with polydimethyl siloxane (PDMS) which we use to fabricate a microfluidic device for ...

科研

生物学

Spinal Cord Electrophysiology

The neonatal mouse spinal cord is a model for studying the development of neural circuitries and locomotor movement. We demonstrate the spinal cord dissection and preparation of recording bath artificial cerebrospinal fluid used for locomotor studies. Once dissected, the spinal cord ventral nerve roots can be attached to a recording electrode to record the electrophysiologic signals of the central pattern generating circuitry within the lumbar cord.

A demonstration of the isolation of neonatal mouse spinal cord for electrophysiologic studies.

脊髓电

The neonatal mouse spinal cord is a model for studying the development of neural circuitries and locomotor movement. We demonstrate the spinal cord ...

Mouse Embryonic Lung Culture, A System to Evaluate the Molecular Mechanisms of Branching

Lung primordial specification as well as branching morphogenesis, and the formation of various pulmonary cell lineages requires a specific interaction of the lung endoderm with its surrounding mesenchyme and mesothelium. Lung mesenchyme has been shown to be the source of inductive signals for lung branching morphogenesis. Epithelial-mesenchymal-mesothelial interactions are also critical to embryonic lung morphogenesis. Early embryonic lung organ culture is a very useful system to study epithelial-mesenchymal interactions. Both epithelial and mesenchymal morphogenesis proceeds under specific conditions that can be readily manipulated in this system (in the absence of maternal influence and blood flow). More importantly this technique can be readily done in a serumless, chemically defined culture media. Gain and loss of function can be achieved using expressed proteins, recombinant viral vectors and/or analysis of transgenic mouse strains, antisense RNA, as well as RNA interference gene knockdown.

Early embryonic lung organ culture is a very useful system to study epithelial-mesenchymal interactions. Both epithelial and mesenchymal morphogenesis proceeds under specific conditions that can be readily manipulated in this system.

小鼠胚胎的龙文化，一个系统来评价分枝的分子机制

Lung primordial specification as well as branching morphogenesis, and the formation of various pulmonary cell lineages requires a specific interaction of ...

Adenovirus-mediated Genetic Removal of Signaling Molecules in Cultured Primary Mouse Embryonic Fibroblasts

The ability to genetically remove specific components of various cell signalling cascades has been an integral tool in modern signal transduction analysis. One particular method to achieve this conditional deletion is via the use of the Cre-loxP system. This method involves flanking the gene of interest with loxP sites, which are specific recognition sequences for the Cre recombinase protein. Exposure of the so-called floxed (flanked by loxP site) DNA to this enzyme results in a Cre-mediated recombination event at the loxP sites, and subsequent excision of the intervening gene3. Several different methods exist to administer Cre recombinase to the site of interest. In this video, we demonstrate the use of an adenovirus containing the Cre recombinase gene to infect primary mouse embryonic fibroblasts (MEFs) obtained from embryos containing a floxed Rac1 allele1. Our rationale for selecting Rac1 MEFs for our experiments is that clear morphological changes can be seen upon deletion of Rac1, due to alterations in the actin cytoskeleton2,5. 72 hours following viral transduction and Cre expression, cells were stained using the actin dye phalloidin and imaged using confocal laser scanning microscopy. It was observed that MEFs which had been exposed to the adeno-Cre virus appeared contracted and elongated in morphology compared to uninfected cells, consistent with previous reports2,5. The adenovirus method of Cre recombinase delivery is advantageous as the adeno-Cre virus is easily available, and gene deletion via Cre in nearly 100% of the cells can be achieved with optimized adenoviral infection.

In this video we use an adenovirus carrying the Cre recombinase gene to infect primary mouse embryonic fibroblasts carrying a floxed Rac1 allele.

腺病毒介导的基因去除信号分子在原代培养的小鼠胚胎成纤维

The ability to genetically remove specific components of various cell signalling cascades has been an integral tool in modern signal transduction ...

Photobleaching Assays (FRAP &amp; FLIP) to Measure Chromatin Protein Dynamics in Living Embryonic Stem Cells

Fluorescence Recovery After Photobleaching (FRAP) and Fluorescence Loss In Photobleaching (FLIP) enable the study of protein dynamics in living cells with good spatial and temporal resolution. Here we describe how to perform FRAP and FLIP assays of chromatin proteins, including H1 and HP1, in mouse embryonic stem (ES) cells. In a FRAP experiment, cells are transfected, either transiently or stably, with a protein of interest fused with the green fluorescent protein (GFP) or derivatives thereof (YFP, CFP, Cherry, etc.). In the transfected, fluorescing cells, an intense focused laser beam bleaches a relatively small region of interest (ROI). The laser wavelength is selected according to the fluorescent protein used for fusion. The laser light irreversibly bleaches the fluorescent signal of molecules in the ROI and, immediately following bleaching, the recovery of the fluorescent signal in the bleached area - mediated by the replacement of the bleached molecules with the unbleached molecules - is monitored using time lapse imaging. The generated fluorescence recovery curves provide information on the protein's mobility. If the fluorescent molecules are immobile, no fluorescence recovery will be observed. In a complementary approach, Fluorescence Loss in Photobleaching (FLIP), the laser beam bleaches the same spot repeatedly and the signal intensity is measured elsewhere in the fluorescing cell. FLIP experiments therefore measure signal decay rather than fluorescence recovery and are useful to determine protein mobility as well as protein shuttling between cellular compartments. Transient binding is a common property of chromatin-associated proteins. Although the major fraction of each chromatin protein is bound to chromatin at any given moment at steady state, the binding is transient and most chromatin proteins have a high turnover on chromatin, with a residence time in the order of seconds. These properties are crucial for generating high plasticity in genome expression1. Photobleaching experiments are therefore particularly useful to determine chromatin plasticity using GFP-fusion versions of chromatin structural proteins, especially in ES cells, where the dynamic exchange of chromatin proteins (including heterochromatin protein 1 (HP1), linker histone H1 and core histones) is higher than in differentiated cells2,3.

Photobleaching Assays FRAP &amp; FLIP to Measure Chromatin Protein Dynamics in Living Embryonic Stem Cells

We describe photobleaching methods including Fluorescence Recovery After Photobleaching (FRAP) and Fluorescence Loss In Photobleaching (FLIP) to monitor chromatin protein dynamics in embryonic stem (ES) cells. Chromatin protein dynamics, which is considered to be one of the means to study chromatin plasticity, is enhanced in pluripotent cells.

漂白实验（FRAP和FLIP）来衡量生活胚胎干细胞的染色质的蛋白质动力学

Fluorescence Recovery After Photobleaching (FRAP) and Fluorescence Loss In Photobleaching (FLIP) enable the study of protein dynamics in living cells with ...

Assaying the Kinase Activity of LRRK2 in vitro

Leucine Rich Repeat Kinase 2 (LRRK2) is a 2527 amino acid member of the ROCO family of proteins, possessing a complex, multidomain structure including a GTPase domain (termed ROC, for Ras of Complex proteins) and a kinase domain1. The discovery in 2004 of mutations in LRRK2 that cause Parkinson's disease (PD) resulted in LRRK2 being the focus of a huge volume of research into its normal function and how the protein goes awry in the disease state2,3. Initial investigations into the function of LRRK2 focused on its enzymatic activities4-6. Although a clear picture has yet to emerge of a consistent alteration in these due to mutations, data from a number of groups has highlighted the importance of the kinase activity of LRRK2 in cell death linked to mutations7,8. Recent publications have reported inhibitors targeting the kinase activity of LRRK2, providing a key experimental tool9-11. In light of these data, it is likely that the enzymatic properties of LRRK2 afford us an important window into the biology of this protein, although whether they are potential drug targets for Parkinson's is open to debate.
A number of different approaches have been used to assay the kinase activity of LRRK2. Initially, assays were carried out using epitope tagged protein overexpressed in mammalian cell lines and immunoprecipitated, with the assays carried out using this protein immobilised on agarose beads4,5,7. Subsequently, purified recombinant fragments of LRRK2 in solution have also been used, for example a GST tagged fragment purified from insect cells containing residues 970 to 2527 of LRRK212. Recently, Dani&euml;ls et al. reported the isolation of full length LRRK2 in solution from human embryonic kidney cells, however this protein is not widely available13. In contrast, the GST fusion truncated form of LRRK2 is commercially available (from Invitrogen, see table 1 for details), and provides a convenient tool for demonstrating an assay for LRRK2 kinase activity. Several different outputs for LRRK2 kinase activity have been reported. Autophosphorylation of LRRK2 itself, phosphorylation of Myelin Basic Protein (MBP) as a generic kinase substrate and phosphorylation of an artificial substrate - dubbed LRRKtide, based upon phosphorylation of threonine 558 in Moesin - have all been used, as have a series of putative physiological substrates including &alpha;-synuclein, Moesin and 4-EBP14-17. The status of these proteins as substrates for LRRK2 remains unclear, and as such the protocol described below will focus on using MBP as a generic substrate, noting the utility of this system to assay LRRK2 kinase activity directed against a range of potential substrates.

Assaying the Kinase Activity of LRRK2 in vitro

Leucine Rich Repeat Kinase 2 is a large multidomain kinase, mutations in which are the most common genetic cause of Parkinson's disease. Analysis of the kinase activity of this protein has proven to be a crucial tool in understanding the biology and dysfunction of this protein. In this paper, in vitro assaying of the kinase activity of LRRK2 and a selection of its mutants is described, providing an experimental system to examine phosphorylation of putative substrates and potential dysfunction of LRRK2 in disease.

含量测定的LRRK2的激酶活性在体外</em

Leucine Rich Repeat Kinase 2 (LRRK2) is a 2527 amino acid member of the ROCO family of proteins, possessing a complex, multidomain structure including a ...

A System for ex vivo Culturing of Embryonic Pancreas

The pancreas controls vital functions of our body, including the production of digestive enzymes and regulation of blood sugar levels1. Although in the past decade many studies have contributed to a solid foundation for understanding pancreatic organogenesis, important gaps persist in our knowledge of early pancreas formation2. A complete understanding of these early events will provide insight into the development of this organ, but also into incurable diseases that target the pancreas, such as diabetes or pancreatic cancer. Finally, this information will generate a blueprint for developing cell-replacement therapies in the context of diabetes.
 During embryogenesis, the pancreas originates from distinct embryonic outgrowths of the dorsal and ventral foregut endoderm at embryonic day (E) 9.5 in the mouse embryo3,4. Both outgrowths evaginate into the surrounding mesenchyme as solid epithelial buds, which undergo proliferation, branching and differentiation to generate a fully mature organ2,5,6. Recent evidences have suggested that growth and differentiation of pancreatic cell lineages, including the insulin-producing &beta;-cells, depends on proper tissue-architecture, epithelial remodeling and cell positioning within the branching pancreatic epithelium7,8. However, how branching morphogenesis occurs and is coordinated with proliferation and differentiation in the pancreas is largely unknown. This is in part due to the fact that current knowledge about these developmental processes has relied almost exclusively on analysis of fixed specimens, while morphogenetic events are highly dynamic.
 Here, we report a method for dissecting and culturing mouse embryonic pancreatic buds ex vivo on glass bottom dishes, which allow direct visualization of the developing pancreas (Figure 1). This culture system is ideally devised for confocal laser scanning microscopy and, in particular, live-cell imaging. Pancreatic explants can be prepared not only from wild-type mouse embryos, but also from genetically engineered mouse strains (e.g. transgenic or knockout), allowing real-time studies of mutant phenotypes. Moreover, this ex vivo culture system is valuable to study the effects of chemical compounds on pancreatic development, enabling to obtain quantitative data about proliferation and growth, elongation, branching, tubulogenesis and differentiation. In conclusion, the development of an ex vivo pancreatic explant culture method combined with high-resolution imaging provides a strong platform for observing morphogenetic and differentiation events as they occur within the developing mouse embryo.

A System for ex vivo Culturing of Embryonic Pancreas

Here, we describe a method for isolation, culture and manipulation of mouse embryonic pancreas. This represents an excellent ex vivo system for studying various aspects of pancreatic development, including morphogenesis, differentiation and growth. Pancreatic bud explants can be cultured for several days and used in a range of different applications, including whole-mount immunofluorescence and live imaging.

一个系统<em&gt;体外</em培养的胚胎胰腺

The pancreas controls vital functions of our body,  including the production of digestive enzymes and regulation of blood sugar  levels1. Although in the ...

Blastomere Explants to Test for Cell Fate Commitment During Embryonic Development

Fate maps, constructed from lineage tracing all of the cells of an embryo, reveal which tissues descend from each cell of the embryo. Although fate maps are very useful for identifying the precursors of an organ and for elucidating the developmental path by which the descendant cells populate that organ in the normal embryo, they do not illustrate the full developmental potential of a precursor cell or identify the mechanisms by which its fate is determined. To test for cell fate commitment, one compares a cell's normal repertoire of descendants in the intact embryo (the fate map) with those expressed after an experimental manipulation. Is the cell's fate fixed (committed) regardless of the surrounding cellular environment, or is it influenced by external factors provided by its neighbors? Using the comprehensive fate maps of the Xenopus embryo, we describe how to identify, isolate and culture single cleavage stage precursors, called blastomeres. This approach allows one to assess whether these early cells are committed to the fate they acquire in their normal environment in the intact embryo, require interactions with their neighboring cells, or can be influenced to express alternate fates if exposed to other types of signals.

The fate of an individual embryonic cell can be influenced by inherited molecules and/or by signals from neighboring cells. Utilizing fate maps of the cleavage stage Xenopus embryo, single blastomeres can be identified for culture in isolation to assess the contributions of inherited molecules versus cell-cell interactions.

卵裂球外植体测试胚胎发育过程中细胞命运的承诺

Fate maps, constructed from lineage tracing all of the cells of an embryo, reveal  which tissues descend from each cell of the embryo.  Although fate maps ...

Mechanical Stimulation-induced Calcium Wave Propagation in Cell Monolayers: The Example of Bovine Corneal Endothelial Cells

Intercellular communication is essential for the coordination of physiological processes between cells in a variety of organs and tissues, including the brain, liver, retina, cochlea and vasculature. In experimental settings, intercellular Ca2+-waves can be elicited by applying a mechanical stimulus to a single cell. This leads to the release of the intracellular signaling molecules IP3 and Ca2+ that initiate the propagation of the Ca2+-wave concentrically from the mechanically stimulated cell to the neighboring cells. The main molecular pathways that control intercellular Ca2+-wave propagation are provided by gap junction channels through the direct transfer of IP3 and by hemichannels through the release of ATP. Identification and characterization of the properties and regulation of different connexin and pannexin isoforms as gap junction channels and hemichannels are allowed by the quantification of the spread of the intercellular Ca2+-wave, siRNA, and the use of inhibitors of gap junction channels and hemichannels. Here, we describe a method to measure intercellular Ca2+-wave in monolayers of primary corneal endothelial cells loaded with Fluo4-AM in response to a controlled and localized mechanical stimulus provoked by an acute, short-lasting deformation of the cell as a result of touching the cell membrane with a micromanipulator-controlled glass micropipette with a tip diameter of less than 1 &mu;m. We also describe the isolation of primary bovine corneal endothelial cells and its use as model system to assess Cx43-hemichannel activity as the driven force for intercellular Ca2+-waves through the release of ATP. Finally, we discuss the use, advantages, limitations and alternatives of this method in the context of gap junction channel and hemichannel research.

Intercellular Ca2+-waves are driven by gap junction channels and hemichannels. Here, we describe a method to measure intercellular Ca2+-waves in cell monolayers in response to a local single-cell mechanical stimulus and its application to investigate the properties and regulation of gap junction channels and hemichannels.

机械刺激引起的钙波传播的细胞单层：牛角膜内皮细胞的例子

Intercellular communication is essential for the coordination of physiological processes between cells in a variety of organs and tissues, including the ...

Genome-wide Mapping of Drug-DNA Interactions in Cells with COSMIC (Crosslinking of Small Molecules to Isolate Chromatin)

The genome is the target of some of the most effective chemotherapeutics, but most of these drugs lack DNA sequence specificity, which leads to dose-limiting toxicity and many adverse side effects. Targeting the genome with sequence-specific small molecules may enable molecules with increased therapeutic index and fewer off-target effects. N-methylpyrrole/N-methylimidazole polyamides are molecules that can be rationally designed to target specific DNA sequences with exquisite precision. And unlike most natural transcription factors, polyamides can bind to methylated and chromatinized DNA without a loss in affinity. The sequence specificity of polyamides has been extensively studied in vitro with cognate site identification (CSI) and with traditional biochemical and biophysical approaches, but the study of polyamide binding to genomic targets in cells remains elusive. Here we report a method, the crosslinking of small molecules to isolate chromatin (COSMIC), that identifies polyamide binding sites across the genome. COSMIC is similar to chromatin immunoprecipitation (ChIP), but differs in two important ways: (1) a photocrosslinker is employed to enable selective, temporally-controlled capture of polyamide binding events, and (2) the biotin affinity handle is used to purify polyamide&#8211;DNA conjugates under semi-denaturing conditions to decrease DNA that is non-covalently bound. COSMIC is a general strategy that can be used to reveal the genome-wide binding events of polyamides and other genome-targeting chemotherapeutic agents.

Genome-wide Mapping of Drug-DNA Interactions in Cells with COSMIC Crosslinking of Small Molecules to Isolate Chromatin

Identifying the direct targets of genome-targeting molecules remains a major challenge. To understand how DNA-binding molecules engage the genome, we developed a method that relies on crosslinking of small molecules to isolate chromatin (COSMIC).