Name: localized rnai and ectopic gene expression in the medicinal leech
Uploaded: 2008-04-17T22:00:00
Description: Watch this Scientific Journal Video about Localized RNAi and Ectopic Gene Expression in the Medicinal Leech at JoVE.com

我们想感谢他的帮助下，在生成的基因结构，基因敲除，并提供不断增长的感觉预测的时间推移电影迈克尔贝克博士。我们还要感谢弗吉尼亚Vandelinder与气动枪的实验技术援助。

粒子可涂用不同的试剂，如双链RNA分子，DNA质粒或染料，。颗粒的类型和直径的选择根据试剂和在该组织的目标细胞：较大的粒子渗透进一步深入，但可能会导致某些组织损伤。 1。 dsRNA的包覆颗粒的制备<ol><li>将冰异丙醇100％。 </li><li>悬浮5毫克的金粒子（S1600ri，贝壳科技有限责任公司;平均直径为1.6微米），在结合缓冲液100毫升。当解决方案已准备就绪，继续下一步。 </li...

<ol>
	<li>Baker, M. W., Macagno, E. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNAi+of+the+receptor+tyrosine+phosphatase+HmLAR2+in+a+single+cell+of+an+intact+leech+embryo+leads+to+growth-cone+collapse.">RNAi of the receptor tyrosine phosphatase HmLAR2 in a single cell of an intact leech embryo leads to growth-cone collapse.</a> Current Biology. 10, 1071-1074 (2000).</li><li>Baker, M. W., Macagno, E. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+Hirudo+medicinalis+DNA+Promoters+for+Targeted+Gene+Expression.">Characterization of Hirudo medicinalis DNA Promoters for Targeted Gene Expression.</a> Journal of Neuroscience Methods. 156, 145-153 (2006).</li><li>Gan, W. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+expression+of+a+leech+netrin+suggests+roles+in+the+formation+of+longitudinal+nerve+tracts+and+in+regional+innervation+of+peripheral+targets.">Cellular expression of a leech netrin suggests roles in the formation of longitudinal nerve tracts and in regional innervation of peripheral targets.</a> Journal of Neurobiology. 40, 103-115 (1999).</li><li>Juven-Gershon, T., Cheng, S., Kadonaga, J. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rational+design+of+a+super+core+promoter+that+enhances+gene+expression.">Rational design of a super core promoter that enhances gene expression.</a> Nature Methods. 3, 917-922 (2006).</li><li>Rinberg, D., Simonnet, C., Groisman, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pneumatic+capillary+gun+for+ballistic+delivery+of+microparticles.">Pneumatic capillary gun for ballistic delivery of microparticles.</a> Applied Physics Letters. 87, 014103 (2005).</li><li>Shefi, O., Simonnet, C., Baker, M. W., Glass, J. R., Macagno, E. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microtargeted+gene+silencing+and+ectopic+expression+in+live+embryos+using+biolistic+delivery+with+a+pneumatic+capillary+gun.">Microtargeted gene silencing and ectopic expression in live embryos using biolistic delivery with a pneumatic capillary gun.</a> Journal of Neuroscience. 26, 6119-6123 (2006).</li></ol>

在这个演示中，我们用毛细管气动枪提供金粒子进入细胞，在一个活的水蛭胚胎的本地化体积小敲，敲除基因。目标区域一般有一个直径150微米的颗粒〜15微米左右的平均穿透深度，这是在0和50微米之间可调。当粒子与双链RNA分子的轴突导向因子netrin涂层，它们造成明显可见netrin的表达，只有在含有颗粒细胞发生击倒。粒子涂在神经细胞中的质粒编码绿色荧光蛋白诱导荧光，当他们停止在其原子核。 我们已...

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<td>gold particles</td>
<td>Other</td>
<td>Seashell Technology, LLC</td>
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<td>binding buffer</td>
<td>Reagent</td>
<td>Seashell Technology, LLC</td>
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<td>silicone rubber </td>
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localized rnai and ectopic gene expression in the medicinal leech

在这个视频中，我们展示的气动毛细血管枪使用的试剂准确的枪法交付到现场组织。我们扰乱基因表达模式在使用过程水蛭胚胎选择的细分，使内部控制未经处理的部分。 毛细管气动枪可用于开放的标本，在发展初期没有达到内部的细胞层。作为本地化的物质引入活组织法，基因枪与枪的交付有几个优点：它是快速，无接触式和非破坏性的。此外，一个单一的毛细血管枪，可用于不同物质的独立交付。配送区域〜50-150微米〜15微米，横向尺寸和延伸，各地的平均穿透深度，这是0和50微米之间可调。这次交付的优点是能够在选定的位置之间的单细胞，通过显微注射和全身击倒通过胞注射或通过遗传方法敲下来中间目标的细胞数量有限。 对于撞倒或敲在轴突导向分子Netrin，这自然是一些中央神经元和腹侧体壁的表达，但不背域的表达，我们提供的双链RNA分子或质粒DNA和体壁中央神经节。此过程包括以下步骤：（一）编制的一个特定的检测实验的设置（调整加快的压力），（二）涂层与双链RNA或DNA分子的颗粒，（三）包覆颗粒装入枪，在一个实验中，（四）两个试剂准备粒子传递，传递到靶组织（体壁或神经节）（V）的包覆颗粒的动物，及（vi）处理的胚胎（免疫，免疫组织化学和神经元标签）以可视化的结果，通常交货后2至3天。 当粒子被涂netrin双链RNA，它们造成明显可见netrin的表达，只有在含有颗粒的细胞（通常情况下，每单元1-2颗粒）发生击倒。粒子涂在神经细胞中的质粒编码EGFP的诱导荧光，当他们停止在其原子核。

Watch this Scientific Journal Video about Localized RNAi and Ectopic Gene Expression in the Medicinal Leech at JoVE.com

Localized RNAi and Ectopic Gene Expression in the Medicinal Leech

在这个视频中，我们展示一个准确的枪法交付到现场组织了一种新型微型基因枪试剂的过程。我们撞倒在水蛭胚胎提供腹侧体壁和神经节单节段双链RNA分子的轴突导向分子Netrin的表达。

本地化的RNAi和药用水蛭异位基因表达

In this video, we show the use of a pneumatic capillary gun for the accurate biolistic delivery of reagents into live tissue. We use the procedure to ...

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