Name: stereotactic injection of microrna-expressing lentiviruses to the mouse hippocampus ca1 region and assessment of the behavioral outcome
Uploaded: 2013-06-10T12:00:00
Description: Watch this Scientific Journal Video about Stereotactic Injection of MicroRNA-expressing Lentiviruses to the Mouse Hippocampus CA1 Region and Assessment of the Behavioral Outcome at JoVE.com

这项研究已经得到了爱德蒙和莉莉·萨夫拉中心脑科学（SB奖学金），以色列科学基金会（批准号：378/11，HS）的遗产遗产生物医学科学合作计划和德国以色列科学基金会研究与发展（GIF）（批准号：1093-32.2/2010 HS）。

1。慢病毒载体的制备<ol><li> HEK-293FT细胞增长到90％汇合。 </li><li>上的天的的变动转染细胞培养基中无血清DMEM辅以用1mM谷氨酰胺和50 mg / ml的青霉素 - 链霉素。 </li><li>共同转染的的细胞与pLKO.1迪普罗载体有质粒的编码为ΔR8.2和VSV-G部分和感兴趣的miRNA，用10微升的1毫克/毫升的聚乙烯亚胺作为载体9。 </li><li>在24小时和48小时后转染，收集打包慢病毒过滤0.45微米的过滤器。 </li><li>利用超速离...

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	<li>Siegel, G., 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=A+functional+screen+implicates+microRNA-138-dependent+regulation+of+the+depalmitoylation+enzyme+APT1+in+dendritic+spine+morphogenesis.">A functional screen implicates microRNA-138-dependent regulation of the depalmitoylation enzyme APT1 in dendritic spine morphogenesis.</a> Nature Cell Biology. 11, 705-716 (2009).</li><li>Jin, P., 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=Biochemical+and+genetic+interaction+between+the+fragile+X+mental+retardation+protein+and+the+microRNA+pathway.">Biochemical and genetic interaction between the fragile X mental retardation protein and the microRNA pathway.</a> Nature Neuroscience. 7, 113-117 (2004).</li><li>Simon, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+microRNA+miR-1+regulates+a+MEF-2-dependent+retrograde+signal+at+neuromuscular+junctions.">The microRNA miR-1 regulates a MEF-2-dependent retrograde signal at neuromuscular junctions.</a> Cell. 133, 903-915 (2008).</li><li>Consiglio, A. <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+gene+therapy+of+metachromatic+leukodystrophy+by+lentiviral+vectors:+correction+of+neuropathology+and+protection+against+learning+impairments+in+affected+mice.">In vivo gene therapy of metachromatic leukodystrophy by lentiviral vectors: correction of neuropathology and protection against learning impairments in affected mice.</a> Nature Medicine. 7, 310-316 (2001).</li><li>Jakobsson, J., Lundberg, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lentiviral+vectors+for+use+in+the+central+nervous+system.">Lentiviral vectors for use in the central nervous system.</a> Molecular Therapy: The Journal of the American Society of Gene Therapy. 13, 484-493 (2006).</li><li>Berson, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cholinergic-associated+loss+of+hnRNP-A/B+in+Alzheimer's+disease+impairs+cortical+splicing+and+cognitive+function+in+mice.">Cholinergic-associated loss of hnRNP-A/B in Alzheimer's disease impairs cortical splicing and cognitive function in mice.</a> EMBO Molecular Medicine. , (2012).</li><li>Haramati, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MicroRNA+as+repressors+of+stress-induced+anxiety:+the+case+of+amygdalar+miR-34.">MicroRNA as repressors of stress-induced anxiety: the case of amygdalar miR-34.</a> The Journal of Neuroscience: The Official Journal of the Society for Neuroscience. 31, 14191-14203 (2011).</li><li>Shaltiel, G., 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=Hippocampal+microRNA-132+mediates+stress-inducible+cognitive+deficits+through+its+acetylcholinesterase+target.">Hippocampal microRNA-132 mediates stress-inducible cognitive deficits through its acetylcholinesterase target.</a> Brain Structure &amp; Function. , 10-1007 (2012).</li><li>Boussif, O., 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=A+versatile+vector+for+gene+and+oligonucleotide+transfer+into+cells+in+culture+and+in+vivo:+polyethylenimine.">A versatile vector for gene and oligonucleotide transfer into cells in culture and in vivo: polyethylenimine.</a> Proceedings of the National Academy of Sciences of the United States of America. 92, 7297-7301 (1995).</li><li>Mendenhall, A., Lesnik, J., Mukherjee, C., Antes, T., Sengupta, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Packaging+HIV-+or+FIV-based+lentivector+expression+constructs+&amp;+transduction+of+VSV-G+pseudotyped+viral+particles.">Packaging HIV- or FIV-based lentivector expression constructs &amp; transduction of VSV-G pseudotyped viral particles.</a> J. 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立体定向注射慢病毒是一个相对快速的方法，无论是向上或向下调节不同的基因和miRNA 在体内评估。主要的替代是一个遗传工程小鼠，这是一个更费时费力的技术，然后慢直喷。此外，向上调节，在慢病毒注射，在成年小鼠发生在一个特定的时间，并且不包括任何可能的泄漏在开发过程中，是最经常的情况下，包括时间控制遗传工程小鼠。不同的基因在神经退行性疾病中的调控改变的动物?...

miRNA在神经系统功能的作用，尤其是最近已经慢病毒注射在几项研究中的挑战。已发现miRNA的维护和再塑造突触结构参数1 2，突触发生和突触重塑和维护3是至关重要的。这些研究有力地表明，miRNA的啮合时，通过多层次的调节作用，在形成和维持神经系统的主要输出，认知功能。立体定向注射慢病毒颗粒可以搜索到特定地区的啮齿类动物的大脑突触形态的改变和神经元的活动，并用于建立过表达的转录4,5的功能意义。义脑区的神经元与miRNA的表达慢病毒直接感染可能受到牵连老化，脑部疾病和神经退行性疾病的研究; miRNA的研究在行为调节6-8的境界是在一个远远低于先进国家，立体注射的miRNA表达慢病毒颗粒，然后通过行为测试等用途可能是有用的。诱导过度或不足表达相当费力的方法涉及遗传工程连锁或基因敲除小鼠。遗传系统可以进一步允许表达的条件和时间的控制（ 例如 ，春节系统CRE-LOX），但很难提供空间特异性注射过程几乎总是有一定量的泄漏。此外，工程程序并不需要手术治疗，可用于在实验室比较好的重现性，但是，他们是慢，需要更多的人力和财力。此外，时间控制的过表达小鼠注射准确得多遗传工程小鼠相比。

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		<col />
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	<tbody>
		<tr height="22">
			<td></td>
			<td></td>
			<td></td>
			<td>Equipment</td>
		</tr>
		<tr height="21">
			<td height="21">Rodent weigh scale</td>
			<td>Burtons (UK)</td>
			<td>115-455</td>
			<td>&nbsp;</td>
		</tr>
		<tr height="24">
			<td height="24">heating pad</td>
			<td>FIRstTechnology</td>
			<td>DCT-25</td>
			<td>&nbsp;</td>
		</tr>
		<tr height="21">
			<td height="21">trimming machine</td>
			<td>Stoelting</td>
			<td>51465</td>
			<td>&nbsp;</td>
		</tr>
		<tr height="21">
			<td height="21">stereotact</td>
			<td>Stoelting</td>
			<td>51730</td>
			<td>&nbsp;</td>
		</tr>
		<tr height="21">
			<td height="21">Scalpel and blades</td>
			<td>Kent scientific</td>
			<td>INS500348</td>
			<td>&nbsp;</td>
		</tr>
		<tr height="21">
			<td height="21">Harland syringe</td>
			<td>Hamilton</td>
			<td>7632-01</td>
			<td>&nbsp;</td>
		</tr>
		<tr height="21">
			<td height="21">driller</td>
			<td>Stoelting</td>
			<td>51449</td>
			<td>&nbsp;</td>
		</tr>
		<tr height="21">
			<td height="21">digital pump</td>
			<td>Harvard apparatus</td>
			<td>704507</td>
			<td>&nbsp;</td>
		</tr>
		<tr height="21">
			<td height="21">Water tank and platform</td>
			<td>Stoelting</td>
			<td>60135</td>
			<td>&nbsp;</td>
		</tr>
	</tbody>
</table>
<table border="0" cellpadding="0" cellspacing="0" width="682">
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			<td></td>
			<td></td>
			<td>Reagents</td>
		</tr>
		<tr height="21">
			<td height="21">ketamine </td>
			<td>Vetoquinol(Lure France)</td>
			<td>3055503</td>
			<td>&nbsp;</td>
		</tr>
		<tr height="21">
			<td height="21">domitor</td>
			<td>Orion pharma</td>
			<td>107140-10</td>
			<td>&nbsp;</td>
		</tr>
		<tr height="21">
			<td height="21">Rimadyl</td>
			<td>Pfizer animal health</td>
			<td>24751</td>
			<td>&nbsp;</td>
		</tr>
		<tr height="118">
			<td height="138" rowspan="2">moisture ointment - Synthomycine 5%</td>
			<td rowspan="2">Rekah Pharmaceutical</td>
			<td rowspan="2">195</td>
			<td rowspan="2"> </td>
		</tr>
		<tr height="20">
		</tr>
		<tr height="21">
			<td height="21">histoacryl</td>
			<td>Braun</td>
			<td>112101</td>
			<td>&nbsp;</td>
		</tr>
		<tr height="21">
			<td height="21">saline</td>
			<td>Sigma Aldrich</td>
			<td>D8662</td>
			<td>&nbsp;</td>
		</tr>
	</tbody>
</table>

stereotactic injection of microrna-expressing lentiviruses to the mouse hippocampus ca1 region and assessment of the behavioral outcome

小分子RNA（miRNA）是监管小单约22个核苷酸长的双链RNA分子可能每个目标众多的mRNA转录和暗淡的整个基因表达通路诱导杀伤性和/或抑制翻译这些目标。一些miRNA的发挥关键作用，在维持神经元的结构和功能，并在更高级别的脑功能，寻求探索这些功能操作水平和方法。在这里，我们提出了一种直接在体内的方法强迫立体定向注射miRNA的编码病毒颗粒在小鼠体内多余的miRNA研究的认知后果。具体来说，目前的协议还包括海马CA1区，这有助于哺乳动物记忆的巩固，学习和应激反应，并提供了一​​个方便的注射部位注入。坐标测量，根据鼠标的前囟门和数字控制病毒的灌注，并保持很慢。注射后，手术伤口是密封的，动物的恢复。相应的靶mRNA编码消音服务慢病毒牵连特定miRNA /靶相互作用负责观察效果，​​天真的小鼠，小鼠注射用生理盐水与“空”的慢病毒载体作为对照组小鼠注射。一个月后注射，动物们检查Morris水迷宫（MWM）评估其导航的学习和记忆能力。 MWM是圆罐，充满了彩色水淹没水面1厘米以下的小平台。周围的坦克的稳态视觉线索允许空间导航（声音和地球磁场也可协助动物在导航）。视频摄像监控，使测量游的路线和时间，找到并达到平台。先教鼠标，安装隐藏的平台，提供了一个逃离强迫游泳，然后测试使用这种转义和f值为，该平台将被删除，探针测试检查，如果鼠标记得它以前的位置。连续几天的反复测试，突出改善性能测试小鼠在更短的延迟，是寻找和挂载平台，更直接的路线到达平台或位置。没有这样的代表减值改善学习记忆和/或焦虑，然后可以专门测试（ 如在高架十字迷宫）。这种方法使验证特定的miRNA在所研究的认知和/或压力相关的进程和目标成绩单。

0.5微升慢病毒注射到小鼠海马CA1区的协议部分中的指示的流量，产生约1毫米的喙尾椎轴受感染的球体，大约0.5mm的内侧 - 外侧和前后的轴（ 图3）。 <img alt="图1" src="/files/ftp_upload/50170/50170fig1.jpg" /> 图1。前囟门点和喷射装置 。 （ 一 ）所示是一个示意图，地图的前囟门（冠状面和矢状面的缝合线交汇）和L...

Watch this Scientific Journal Video about Stereotactic Injection of MicroRNA-expressing Lentiviruses to the Mouse Hippocampus CA1 Region and Assessment of the Behavioral Outcome at JoVE.com

Stereotactic Injection of MicroRNA-expressing Lentiviruses to the Mouse Hippocampus CA1 Region and Assessment of the Behavioral Outcome (Video) | JoVE

小分子RNA在大脑的结构和功能有显着的作用。在这里，我们描述了一种方法来执行海马miRNA的过表达一个精心设计的miRNA表达慢病毒采用立体定向注射。这种方法可以作为一个相对快速的方式评估<em&gt;在体内</em&gt;过表达miRNA在特定的大脑区域的影响。

立体定向注射的microRNA表达慢病毒的小鼠海马CA1区和行为结果的评估

MicroRNAs (miRNAs) are small regulatory single-stranded RNA molecules around 22 nucleotides long that may each target numerous mRNA transcripts and dim an ...

Research

JoVE Journal

Behavior

Stereotactic Injection of MicroRNA-expressing Lentiviruses to the Mouse Hippocampus CA1 Region and Assessment of the Behavioral Outcome

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