Name: assaying the ability of diffusible signaling molecules to reorient embryonic spinal commissural axons
Uploaded: 2010-03-08T16:41:00
Description: Watch this Scientific Journal Video about Assaying the Ability of Diffusible Signaling Molecules to Reorient Embryonic Spinal Commissural Axons at JoVE.com

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?...

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	<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 ...

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