We thank Souvik Maiti for help in designing the antagomirs. We also acknowledge Rangeetha J. Naik, Rakesh Dey, and Bijay Pattnaik for their help with experimental methods. This work was funded by the Council of Scientific and Industrial Research (BSC0123). HS, MV and RR acknowledge fellowship from the Council of Scientific and Industrial Research, India. MAS acknowledge fellowship from the University Grants Commission, India.

注:所有包括动物受试者的程序已经被批准机构动物伦理委员会（原​​子能委员会）的基因组学研究所与整合生物学，新德里（IGIB / AEC / 10/2013年）。这个协议是专为中的miR-29的脑和击倒靶向递送RNA拮抗剂-29的调节。 1. RNA拮抗剂设计策略<ol><li>检索来自miRBase 11成熟miRNA序列<a href="http://www.mirbase.org/">（http://www.mirbase.org/）。</a> </li><li>检索相关的miRNA家族成员使用的miRBase记录的"基因家族"链接序列 <a href="http://www.mirbase.org/cgi-bin/mirna_summary.pl?fam=MIPF0000009">（http://www.mirbase.org/cgi-bin/mirna_summary.pl?fam=MIPF0000009）。</a> </li><li>反向互补序列<a href="http://www.bioinformatics.org/sms/rev_comp.html">:http://www.bioinformatics.org/sms/rev_comp.html</a>提供了一个方便的工具反向互补序列。 </li><li>使用以下准则标记由LNA修饰的位置: <ol><li>选择胸腺嘧啶残基放置3-4个碱基分开用于LNA修饰，因为LNA嘧啶是比嘌呤12更稳定。 </li><li>选择胞嘧啶残基3-4残余物中分离，如果前面的步骤产生不到五年的修改。 </li><li>优先的五个修改到微小RNA拮抗剂的5&#39;端，因为这可避免的种子序列。 </li></ol></li></ol> 2. RNA拮抗剂，神经肽复合制剂注意:这是在协议中最关键的步骤，它需要标准化。标准化任何荧光标记的寡核苷酸（FLO）可以代替RNA拮抗剂9的要使用的协议。肽应该是高纯度的（HPLC级，纯度98％）.Sequences和电荷肽和RNA拮抗剂的列于表1。 <ol><li>确定莫数量微小RNA拮抗剂的莱斯基于上微小RNA拮抗剂的鼠标，其毒性和重量摩尔电荷注入。基于RNA拮抗剂的摩尔电荷比计算肽的摩尔数被注入:肽。 注:规范RNA拮抗剂的摩尔电荷比:肽毒性和有效的传递在小鼠大脑。使用荧光标记的寡核苷酸的不同摩尔电荷​​比:肽进行标准化。我们发现，微小RNA拮抗剂-29和对照均在4microgram /克体重的小鼠的毒性。微小RNA拮抗剂-29和控制被用于在2microgram /克体重，但这种有效浓度可能随每个微RNA。 </li><li>稀释在从储备液用无菌10％D-葡萄糖至所需的最终浓度分离微量离心管的肽和RNA拮抗剂作为计算在步骤1。 </li><li>保持离心管，以中等涡旋速度在涡流肽的解决方案。 </li><li>加入1/3 RD的一对ntagomir溶液到肽溶液管，慢慢逐滴，而这是在涡旋混合器上充分混合。 </li><li>继续解决方案上的旋涡混合下1分钟，然后让混合物静置1分钟。 </li><li>重复步骤4和5两个次混合在同一离心管将肽溶液的其余RNA拮抗剂溶液。缓慢加入为的单分散复合物能有效地转染的形成至关重要。快速加入可导致配合物及其沉淀的聚集。 </li><li>孵育复为在室温30分钟无涡流。在孵化的这段时间，使其恢复小鼠到注射将执行了实验室。 </li></ol> 3.尾静脉注射的RNA拮抗剂，神经肽复合物<ol><li>为了抑制动物，将鼠标在适当大小的限制部分或decapicone。 </li><li>使用棉签公关清洁尾部的表面电子浸泡在温水（约40℃）。这将促使血管扩张，增加静脉的知名度。 </li><li>接近尾用小体积（0.5 - 1.0毫升）胰岛素注射器15-20°角。注意不要引入任何空气进入注射器。开始在尾的远端部分。注入复杂。取出针，直接涂抹防腐剂棉球消毒注射部位（约5-10秒），以制止任何出血。 </li><li>更换鼠标在组的3-5在独立通风笼，用玉米芯和纸巾的富集。不要让未注射及注射的小鼠在同一个笼子里。 </li></ol> 4.行为测定注意:保持3-4小时的时间间隔中注入和行为测定之间，以允许注射后恢复。把鼠标一个安静的房间。在此期间，水土不服，不要打扰动物。 <ol><li>逐渐适应鼠标到新空间30分钟，然后启动以下行为测定。保持10分钟的间隙中的每个行为测定之间。 </li><li>后肢夹紧: 注:此试验表明运动功能障碍和神经系统损伤。 <ol><li>握住尾巴的基地附近在清晰的背景拿起鼠标轻轻。 </li><li>检查后肢姿势10-15秒。野生型小鼠扩张部分后肢离开腹部，而一个共济失调小鼠趋于缩回的一个或两个后肢朝向腹部的时间超过50％的悬浮13。 </li><li>返回鼠标笼子</li></ol></li><li>礁测试: <ol><li>提起鼠标轻轻握住尾巴，并保持在一个笼子里的窗台。 </li><li>观察小鼠专门在笼子的一角。野生型小鼠可以很容易地通过弯道，而不必担心，失去了平衡。共济失调鼠标失去平衡，冻结或抖动而沿着笼窗台，在角落走。 </li></ol></li><l我&gt;鼠标足迹分析: 注:此法保持吸收片准备就绪，在一个狭窄的跑道地板（〜70厘米长，约5厘米宽〜5厘米高的围墙。） <ol><li>将鼠标轻轻地用一只手，并用刷子上的后肢适用的油墨。 </li><li>上放置的一个狭窄的跑道的地板的吸收性片材。 </li><li>允许小鼠行走或从一端到另一辗过在一条直线上的吸收性片材在跑道。 </li><li>与使用新鲜吸收性片材各小鼠重复该过程两次以上。 </li><li>测量在前进移动两个连续步骤之间的距离。不包括第一和最后几个脚印，其中动物是刚刚启动，并完成它的运行，分别。 </li></ol></li></ol>

<ol>
	<li>Roshan, R., Ghosh, T., Scaria, V., Pillai, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MicroRNAs:+novel+therapeutic+targets+in+neurodegenerative+diseases.">MicroRNAs: novel therapeutic targets in neurodegenerative diseases.</a> Drug discovery today. 14, 1123-1129 (2009).</li><li>Maes, O. C., Chertkow, H. M., Wang, E., Schipper, H. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MicroRNA:+Implications+for+Alzheimer+Disease+and+other+Human+CNS+Disorders.">MicroRNA: Implications for Alzheimer Disease and other Human CNS Disorders.</a> Current Genomics. 10, 154-168 (2009).</li><li>Soifer, H. S., Rossi, J. J., Saetrom, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MicroRNAs+in+Disease+and+Potential+Therapeutic+Applications.">MicroRNAs in Disease and Potential Therapeutic Applications.</a> Mol Ther. 15, 2070-2079 (2007).</li><li>Bader, A. G., Brown, D., Winkler, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Promise+of+MicroRNA+Replacement+Therapy.">The Promise of MicroRNA Replacement Therapy.</a> Cancer research. 70, 7027-7030 (2010).</li><li>Stenvang, J., Petri, A., Lindow, M., Obad, S., Kauppinen, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inhibition+of+microRNA+function+by+antimiR+oligonucleotides.">Inhibition of microRNA function by antimiR oligonucleotides.</a> Silence. 3, 1-17 (2012).</li><li>Trang, 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=Systemic+Delivery+of+Tumor+Suppressor+microRNA+Mimics+Using+a+Neutral+Lipid+Emulsion+Inhibits+Lung+Tumors+in+Mice.">Systemic Delivery of Tumor Suppressor microRNA Mimics Using a Neutral Lipid Emulsion Inhibits Lung Tumors in Mice.</a> Molecular Therapy. 19, 1116-1122 (2011).</li><li>Barbash, S., Hanin, G., Soreq, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stereotactic+Injection+of+MicroRNA-expressing+Lentiviruses+to+the+Mouse+Hippocampus+CA1+Region+and+Assessment+of+the+Behavioral+Outcome.">Stereotactic Injection of MicroRNA-expressing Lentiviruses to the Mouse Hippocampus CA1 Region and Assessment of the Behavioral Outcome.</a> J Vis Exp. (76), e50170 (2013).</li><li>Kumar, 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=Transvascular+delivery+of+small+interfering+RNA+to+the+central+nervous+system.">Transvascular delivery of small interfering RNA to the central nervous system.</a> Nature. 448, 39-43 (2007).</li><li>Roshan, R., 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=Brain-specific+knockdown+of+miR-29+results+in+neuronal+cell+death+and+ataxia+in+mice.">Brain-specific knockdown of miR-29 results in neuronal cell death and ataxia in mice.</a> RNA. 20, 1287-1297 (2014).</li><li>Kaur, H., Wengel, J., Maiti, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thermodynamics+of+DNA&#8722;RNA+Heteroduplex+Formation:+Effects+of+Locked+Nucleic+Acid+Nucleotides+Incorporated+into+the+DNA+Strand.">Thermodynamics of DNA&#8722;RNA Heteroduplex Formation: Effects of Locked Nucleic Acid Nucleotides Incorporated into the DNA Strand.</a> Biochemistry. 47, 1218-1227 (2008).</li><li>Griffiths-Jones, S., Grocock, R. J., Van Dongen, S., Bateman, A., Enright, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=miRBase:+microRNA+sequences,+targets+and+gene+nomenclature.">miRBase: microRNA sequences, targets and gene nomenclature.</a> Nucleic Acids Research. 34, D140-D144 (2006).</li><li>Kaur, H., Babu, B. R., Maiti, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Perspectives+on+Chemistry+and+Therapeutic+Applications+of+Locked+Nucleic+Acid+(LNA).">Perspectives on Chemistry and Therapeutic Applications of Locked Nucleic Acid (LNA).</a> Chemical Reviews. 107, 4672-4697 (2007).</li><li>Guyenet, S. J., 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+Simple+Composite+Phenotype+Scoring+System+for+Evaluating+Mouse+Models+of+Cerebellar+Ataxia.">A Simple Composite Phenotype Scoring System for Evaluating Mouse Models of Cerebellar Ataxia.</a> J. Vis. Exp. (39), (2010).</li><li>Bergen, J. M., Park, I. -. K., Horner, P. J., Pun, S. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nonviral+Approaches+for+Neuronal+Delivery+of+Nucleic+Acids.">Nonviral Approaches for Neuronal Delivery of Nucleic Acids.</a> Pharmaceutical Research. 25, 983-998 (2008).</li><li>Zou, L. -. L., Ma, J. -. L., Wang, T., Yang, T. -. B., Liu, C. -. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell-Penetrating+Peptide-Mediated+Therapeutic+Molecule+Delivery+into+the+Central+Nervous+System.">Cell-Penetrating Peptide-Mediated Therapeutic Molecule Delivery into the Central Nervous System.</a> Current Neuropharmacology. 11, 197-208 (2013).</li><li>Hwang, D. W., 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+brain-targeted+rabies+virus+glycoprotein-disulfide+linked+PEI+nanocarrier+for+delivery+of+neurogenic+microRNA.">A brain-targeted rabies virus glycoprotein-disulfide linked PEI nanocarrier for delivery of neurogenic microRNA.</a> Biomaterials. 32, 4968-4975 (2011).</li></ol>

The authors have nothing to disclose.

Here we demonstrate a widely accessible methodology to study the effects of miRNA modulation. Currently, most attempts at in vivo characterization of miRNA functions involve the creation of knockout mice or a transgenic that expresses a miRNA sponge. Most miRNAs, even the cell type specific ones are expressed in more than one organ. For instance, miRNAs initially thought to be specific to the hematopoietic system are also expressed in the brain, due to the presence of microglia. Thus even a cell type specifi...

微小RNA已成为新的治疗目标，因为它们在基因表达和直接的证据参与疾病的调节普遍作用。 miRNA是正在积极探讨了他们的潜力作为药物靶点1,2。另外，在miRNA表达的改变与几种疾病3和仿真这些变化由miRNA表达的人工扰动的相关联的可用于研究参与疾病表现的细胞途径。 miRNA的靶向药物组织特异性递送目前是miRNA的基础的药物发展的一个重大挑战。拮抗和miRNA模仿是有希望的代理商扰动miRNA水平4-6。但是，特特点增强了它们的特异性和功效必须掺入RNA拮抗剂的设计中，可以使用之前在体内扰动miRNA表达。 微RNA是特别相关<html>在目前无法治愈的神经变性疾病和神经发育疾病的目标。血 - 脑屏障构成限制拮抗在大脑的输送。立体定向注射广泛用于啮齿动物模型，提供分子的特定位置在大脑7。它需要技巧，仪器仪表和时间大量的投资。立体定向注射是侵入性的，包括外科手术，造成至少轻微伤害和被限制在本地投递。同时优先使用靶向神经元的使用细胞穿透肽的可以应对这些限制，因为它们可以通过跨血管途径给药，但违反血脑屏障。这种由狂犬病毒糖蛋白（RVG）衍生肽，以前是用来提供的siRNA对乙型脑炎病毒在小鼠8。我们发现，使用的肽为微小RNA拮抗剂递送中，miRNA可有效在小鼠大脑9撞倒。 内容】"&gt;的miRNA的第二个主要挑战击倒源于miRNA的小尺寸和密切相关的序列同种型的存在。我们采取的示例的mmu-的miR-29家族，包括三个密切相关的同种型中，miR-29a的，b和c。RNA拮抗剂一般也沿着主链修饰以增加它们的稳定性和使它们耐受核酸酶的攻击。锁核酸（LNA的）提供，它们提高的热稳定性，甚至进一步的优点导致了超越目标降解空间位阻10。所有沿主干引入的修改可能是有效的，但昂贵的。前面我们已经看到，超过最优数量的修改不能进一步提高疗效。因此，RNA拮抗剂的设计涉及到RNA拮抗剂的优化改造。 到复杂的微小RNA拮抗剂与亲神经肽，带电七溴到壬精氨酸扩展非共价地被使用。 D型精氨酸残基被使用的，因为它们赋予更高的稳定性，因为它们不被蛋白酶敏感的切割。七溴到壬精氨酸延伸充当有效的细胞渗透剂，虽然它们不赋予细胞类型特异性。通过共价连接的RVG肽与壬精氨酸连接体，亲神经，产生细胞穿透肽。肽的正电荷的残基带负电荷的核酸骨架相互作用，以形成复合物。这些复合物可以用于有效转染的DNA或RNA导入培养的细胞和体内进入组织。

<table><tbody><tr><td>Vortex</td><td></td><td></td><td></td></tr><tr><td>Restrainer or Decapicone</td><td></td><td></td><td></td></tr><tr><td>Narrow runway</td><td></td><td></td><td>~70-cm-long, ~5-cm-wide with ~5-cm-high walls.</td></tr><tr><td>Reagents</td><td></td><td></td><td></td></tr><tr><td>Fluorescently labelled oligonucleotides (siGLO)</td><td>GE Healthcare Dharmacon INC</td><td>D0016300120</td><td></td></tr><tr><td>10% sterile D-glucose</td><td></td><td></td><td></td></tr><tr><td>Antagomir-29</td><td>Exiqon</td><td>custom synthesis</td><td></td></tr><tr><td>Antagomir-control</td><td>Exiqon</td><td>custom synthesis</td><td></td></tr><tr><td>Neuropeptide RVG</td><td>G.L.Biochem (Shanghai) Ltd.</td><td>custom synthesis</td><td>&gt;98% purity</td></tr><tr><td>Neuropeptide RVM</td><td>G.L.Biochem (Shanghai) Ltd.</td><td>custom synthesis</td><td>&gt;98% purity</td></tr><tr><td>Other</td><td></td><td></td><td></td></tr><tr><td>Cotton</td><td></td><td></td><td></td></tr><tr><td>Warm water</td><td></td><td></td><td></td></tr><tr><td>Insulin syringes</td><td></td><td></td><td></td></tr><tr><td>Absorbent sheets</td><td></td><td></td><td></td></tr><tr><td>Ink</td><td></td><td></td><td></td></tr><tr><td>Brush</td><td></td><td></td><td></td></tr><tr><td>Antiseptic</td><td></td><td></td><td></td></tr></tbody></table>

a simple alternative to stereotactic injection for brain specific knockdown of mirna

MicroRNAs (miRNAs) are key regulators of gene expression. In the brain, vital processes like neurodevelopment and neuronal functions depend on the correct expression of microRNAs. Perturbation of microRNAs in the brain can be used to model neurodegenerative diseases by modulating neuronal cell death. Currently, stereotactic injection is used to deliver miRNA knockdown agents to specific location in the brain. Here, we discuss strategies to design antagomirs against miRNA with locked nucleotide modifications (LNA). Subsequently describe a method for brain specific delivery of antagomirs, uniformly across different regions of the brain. This method is simple and widely applicable since it overcomes the surgery, associated injury and limitation of local delivery in stereotactic injections. We prepared a complex of neurotropic, cell-penetrating peptide Rabies Virus Glycoprotein (RVG) with antagomir against miRNA-29 and injected through tail vein, to specifically deliver in the brain. The antagomir design incorporated features that allow specific targeting of the miRNA and formation of non-covalent complexes with the peptide. The knock-down of the miRNA in neuronal cells, resulted in apoptotic cell death and associated behavioural defects. Thus, the method can be used for acute models of neuro-degeneration through the perturbation of miRNAs.

用这里介绍的方法，50microgram荧光标记的寡核苷酸（FLO）和〜1:15的摩尔电荷比850microgram RVG肽的复合物:制备并通过尾静脉注射一次（FLO肽）。非嗜神经狂犬病毒矩阵（RVM）肽和FLO的复杂用作传送控制。第二天，将小鼠脑和肝中分离和单细胞悬浮液制备。将细胞在显微镜下观察到绿色荧光。 FLO-RVG络合物成功传递与一天中的时间最短，并检测为绿色荧光在脑细胞而不是在肝脏（图2B，2D），同时...

Watch this Scientific Journal Video about A Simple Alternative to Stereotactic Injection for Brain Specific Knockdown of miRNA at JoVE.com

A Simple Alternative to Stereotactic Injection for Brain Specific Knockdown of miRNA

MicroRNAs play crucial roles in the brain and are potential targets for modeling neuro-degeneration. However, perturbing miRNA levels is challenging due to the short length of miRNA and inaccessibility of the brain tissue. This video presents a method for antagomir design and brain specific delivery using a neuropeptide in mice.

一个简单的替代，以立体定向注射miRNA的脑特异性敲除

MicroRNAs (miRNAs) are key regulators of gene expression.  In the brain, vital processes like neurodevelopment and neuronal functions depend on the ...

a-simple-alternative-to-stereotactic-injection-for-brain-specific

Research

JoVE Journal

Neuroscience

12.6K 次观看.  CSIR-Institute of Genomics and Integrative Biology, New Delhi, India. MicroRNAs play crucial roles in the brain and are potential targets for modeling neuro-degeneration. However, perturbing miRNA levels is challenging due to the short length of miRNA and inaccessibility of the brain tissue. This video presents a method for antagomir design and brain specific delivery using a neuropeptide in mice.

视频: A Simple Alternative to Stereotactic Injection for Brain Specific Knockdown of miRNA

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

MicroRNAs (miRNAs) are small regulatory single-stranded RNA molecules around 22 nucleotides long that may each target numerous mRNA transcripts and dim an entire gene expression pathway by inducing destruction and/or inhibiting translation of these targets. Several miRNAs play key roles in maintaining neuronal structure and function and in higher-level brain functions, and methods are sought for manipulating their levels for exploring these functions. Here, we present a direct in vivo method for examining the cognitive consequences of enforced miRNAs excess in mice by stereotactic injection of miRNA-encoding virus particles. Specifically, the current protocol involves injection into the hippocampal CA1 region, which contributes to mammalian memory consolidation, learning, and stress responses, and offers a convenient injection site. The coordinates are measured according to the mouse bregma and virus perfusion is digitally controlled and kept very slow. After injection, the surgery wound is sealed and the animals recover. Lentiviruses encoding silencers of the corresponding mRNA targets serve to implicate the specific miRNA/target interaction responsible for the observed effect, with na&iuml;ve mice, mice injected with saline and mice injected with "empty" lentivirus vectors as controls. One month post-injection, the animals are examined in the Morris Water Maze (MWM) for assessing their navigation learning and memory abilities. The MWM is a round tank filled with colored water with a small platform submerged 1 cm below the water surface. Steady visual cues around the tank allow for spatial navigation (sound and the earth's magnetic field may also assist the animals in navigating). Video camera monitoring enables measuring the route of swim and the time to find and amount the platform. The mouse is first taught that mounting the hidden platform offers an escape from the enforced swimming; it is then tested for using this escape and finally, the platform is removed and probe tests examine if the mouse remembers its previous location. Repeated tests over several consecutive days highlight improved performance of tested mice at shorter latencies to find and mount the platform, and as more direct routes to reach the platform or its location. Failure to show such improvement represents impaired learning and memory and/or anxiety, which may then be tested specifically (e.g. in the elevated plus maze). This approach enables validation of specific miRNAs and target transcripts in the studied cognitive and/or stress-related processes.

MicroRNAs have significant roles in brain structure and function. Here we describe a method to enforce hippocampal miRNA over-expression using stereotactic injection of an engineered miRNA-expressing lentivirus. This approach can serve as a relatively rapid way to assess the in vivo effects of over-expressed miRNAs in specific brain regions.

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 ...

科研

行为学

Biotin-based Pulldown Assay to Validate mRNA Targets of Cellular miRNAs

MicroRNAs (miRNAs) are a class of small noncoding RNAs that post-transcriptionally regulate cellular gene expression. MiRNAs bind to the 3&#39; untranslated region (UTR) of target mRNA to inhibit protein translation or in some instances cause mRNA degradation. The binding of the miRNA to the 3&#39; UTR of the target mRNA is mediated by a 2&#8211;8 nucleotide seed sequence at the 5&#39; end of miRNA. While the role of miRNAs as cellular regulatory molecules is well established, identification of the target mRNAs with functional relevance remains a challenge. Bioinformatic tools have been employed to predict sequences within the 3&#39; UTR of mRNAs as potential targets for miRNA binding. These tools have also been utilized to determine the evolutionary conservation of such sequences among related species in an attempt to predict functional role. However, these computational methods often generate false positive results and are limited to predicting canonical interaction between miRNA and mRNA. Therefore, experimental procedures that measure direct binding of miRNA to its mRNA target are necessary to establish functional interaction. In this report, we describe a sensitive method for validating direct interaction between the cellular miRNA miR-125b and the 3&#39; UTR of PARP-1 mRNA. We elaborate a protocol in which synthetic biotinylated-miRNA mimics were transfected into mammalian cells and the miRNA-mRNA complex in the cellular lysate was pulled down with streptavidin-coated magnetic beads. Finally, the target mRNA in the pulled-down nucleic acid complex was quantified using a qPCR-based strategy.

This report describes a fast and reliable method for validating mRNA targets of cellular miRNAs. The method uses synthetic biotinylated Locked Nucleic Acid (LNA)-based miRNA mimics to capture target mRNA. Subsequently, streptavidin-coated magnetic beads are employed to pulldown the target mRNA for quantification by qPCR polymerase chain reaction.

基于生物素的下拉法对细胞 miRNAs mRNA 靶点的验证

MicroRNAs (miRNAs) are a class of small noncoding RNAs that post-transcriptionally regulate cellular gene expression. MiRNAs bind to the 3&#39; ...

遗传学

Characterization of Functionally Associated miRNAs in Glioblastoma and their Engineering into Artificial Clusters for Gene Therapy

The biological relevance of microRNAs (miRNAs) in health and disease significantly relies on specific combinations of many simultaneously deregulated miRNAs rather than the action of a single miRNA. The characterization of these specific miRNAs modules is a fundamental step in maximizing their use in therapy. This is extremely relevant because their combinatorial attributes can be practically exploited. Described here is a method to define a specific miRNA signature relevant to the control of oncogenic chromatin repressors in glioblastoma. The approach first defines a general group of miRNAs that are deregulated in tumors in comparison to normal tissue. The analysis is further refined by differential culture conditions, underscoring a subgroup of miRNAs that are co-expressed simultaneously during specific cellular states. Finally, the miRNAs that satisfy these filters are combined into an artificial polycistronic transgenes, which is based on a scaffold of naturally existing miRNA clusters genes, then used for overexpression of these miRNA modules into receiving cells.

Described here is a protocol for characterizing modules of biologically synergistic miRNAs and their assembly into short transgenes, which allows simultaneous overexpression for gene therapy applications.

胶质母细胞瘤中功能相关miRNA的特性及其工程在基因治疗的人工簇中

The biological relevance of microRNAs (miRNAs) in health and disease significantly relies on specific combinations of many simultaneously deregulated ...

In ovo Electroporation of miRNA-based Plasmids in the Developing Neural Tube and Assessment of Phenotypes by DiI Injection in Open-book Preparations

Commissural dI1 neurons have been extensively studied to elucidate the mechanisms underlying axon guidance during development1,2. These neurons are located in the dorsal spinal cord and send their axons along stereotyped trajectories. Commissural axons initially project ventrally towards and then across the floorplate. After crossing the midline, these axons make a sharp rostral turn and project longitudinally towards the brain. Each of these steps is regulated by the coordinated activities of attractive and repulsive guidance cues. The correct interpretation of these cues is crucial to the guidance of axons along their demarcated pathway. Thus, the physiological contribution of a particular molecule to commissural axon guidance is ideally investigated in the context of the living embryo. Accordingly, gene knockdown in vivo must be precisely controlled in order to carefully distinguish axon guidance activities of genes that may play multiple roles during development.
Here, we describe a method to knockdown gene expression in the chicken neural tube in a cell type-specific, traceable manner. We use novel plasmid vectors3 harboring cell type-specific promoters/enhancers that drive the expression of a fluorescent protein marker, followed directly by a miR30-RNAi transcript4 (located within the 3'-UTR of the cDNA encoding the fluorescent protein) (Figure 1). When electroporated into the developing neural tube, these vectors elicit efficient downregulation of gene expression and express bright fluorescent marker proteins to enable direct tracing of the cells experiencing knockdown3. Mixing different RNAi vectors prior to electroporation allows the simultaneous knockdown of two or more genes in independent regions of the spinal cord. This permits complex cellular and molecular interactions to be examined during development, in a manner that is fast, simple, precise and inexpensive. In combination with DiI tracing of commissural axon trajectories in open-book preparations5, this method is a useful tool for in vivo studies of the cellular and molecular mechanisms of commissural axon growth and guidance. In principle, any promoter/enhancer could be used, potentially making the technique more widely applicable for in vivo studies of gene function during development6.
This video first demonstrates how to handle and window eggs, the injection of DNA plasmids into the neural tube and the electroporation procedure. To investigate commissural axon guidance, the spinal cord is removed from the embryo as an open-book preparation, fixed, and injected with DiI to enable axon pathways to be traced. The spinal cord is mounted between coverslips and visualized using confocal microscopy.

In ovo Electroporation of miRNA-based Plasmids in the Developing Neural Tube and Assessment of Phenotypes by DiI Injection in Open-book Preparations

A method by which gene expression in the neural tube can be downregulated in a cell type-specific, traceable manner is described. We demonstrate how in ovo electroporation of microRNA-based plasmids that elicit spatiotemporally controlled RNA interference can be used to investigate commissural axon guidance in the developing neural tube.

基于miRNA的质粒在发展中的神经管和表型的评估DiI荧光注射开卷准备在卵的电

Commissural dI1 neurons have been extensively studied to elucidate the mechanisms underlying axon guidance during development1,2. These neurons are ...

神经科学

Design and Microinjection of Morpholino Antisense Oligonucleotides and mRNA into Zebrafish Embryos to Elucidate Specific Gene Function in Heart Development

The morpholino oligomer-based knockdown system has been used to identify the function of various gene products through loss or reduced expression. Morpholinos (MOs) have the advantage in biological stability over DNA oligos because they are not susceptible to enzymatic degradation. For optimal effectiveness, MOs are injected into 1-4 cell stage embryos. The temporal efficacy of knockdown is variable, but MOs are believed to lose their effects due to dilution eventually. Morpholino dilution and injection amount should be closely controlled to minimize the occurrence of off-target effects while maintaining on-target efficacy. Additional complementary tools, such as CRISPR/Cas9 should be performed against the target gene of interest to generate mutant lines and to confirm the morphant phenotype with these lines. This article will demonstrate how to design, prepare, and microinject a translation-blocking morpholino against hand2 into the yolk of 1-4 cell stage zebrafish embryos to knockdown hand2 function and rescue these "morphants" by co-injection of mRNA encoding the corresponding cDNA. Subsequently, the efficacy of the morpholino microinjections is assessed by first verifying the presence of morpholino in the yolk (co-injected with phenol red) and then by phenotypic analysis. Moreover, cardiac functional analysis to test for knockdown efficacy will be discussed. Finally, assessing the effect of morpholino-induced blockage of gene translation via western blotting will be explained.

<meta charset="utf8"></head><body>设计吗啉反义寡核苷酸和 mRNA 并将其显微注射到斑马鱼胚胎中，以阐明心脏发育中的特定基因功能

The present protocol describes designing, preparing, and microinjecting a translational-blocking morpholino against a representative cardiac gene; Heart And Neural Crest Derivatives Expressed2 (hand2) into the yolk of newly fertilized zebrafish embryos to knock down gene function. It also shows a transient rescue of these "morphants" by co-injection of mRNA encoding this gene product.

设计吗啉反义寡核苷酸和 mRNA 并将其显微注射到斑马鱼胚胎中，以阐明心脏发育中的特定基因功能

The morpholino oligomer-based knockdown system has been used to identify the function of various gene products through loss or reduced expression.  ...

发育生物学

Genome-wide Screen for miRNA Targets Using the MISSION Target ID Library

The Target ID Library is designed to assist in discovery and identification of microRNA (miRNA) targets. The Target ID Library is a plasmid-based, genome-wide cDNA library cloned into the 3'UTR downstream from the dual-selection fusion protein, thymidine kinase-zeocin (TKzeo). The first round of selection is for stable transformants, followed with introduction of a miRNA of interest, and finally, selecting for cDNAs containing the miRNA's target. Selected cDNAs are identified by sequencing (see Figure 1-3 for Target ID Library Workflow and details).
To ensure broad coverage of the human transcriptome, Target ID Library cDNAs were generated via oligo-dT priming using a pool of total RNA prepared from multiple human tissues and cell lines. Resulting cDNA range from 0.5 to 4 kb, with an average size of 1.2 kb, and were cloned into the p3&#900;TKzeo dual-selection plasmid (see Figure 4 for plasmid map). The gene targets represented in the library can be found on the Sigma-Aldrich webpage. Results from Illumina sequencing (Table 3), show that the library includes 16,922 of the 21,518 unique genes in UCSC RefGene (79%), or 14,000 genes with 10 or more reads (66%).

The Target ID Library is a plasmid-based, genome-wide collection of cloned cDNA used to identify miRNA targets. Here we demonstrate its use and application.

miRNA的目标使用的任务目标ID库的全基因组屏幕

The Target ID Library is designed to assist in discovery and identification of microRNA (miRNA) targets. The Target ID Library is a plasmid-based, ...

生物学

Identifying Targets of Human microRNAs with the LightSwitch Luciferase Assay System using 3'UTR-reporter Constructs and a microRNA Mimic in Adherent Cells

MicroRNAs (miRNAs) are important regulators of gene expression and play a role in many biological processes. More than 700 human miRNAs have been identified so far with each having up to hundreds of unique target mRNAs. Computational tools, expression and proteomics assays, and chromatin-immunoprecipitation-based techniques provide important clues for identifying mRNAs that are direct targets of a particular miRNA. In addition, 3'UTR-reporter assays have become an important component of thorough miRNA target studies because they provide functional evidence for and quantitate the effects of specific miRNA-3'UTR interactions in a cell-based system. To enable more researchers to leverage 3'UTR-reporter assays and to support the scale-up of such assays to high-throughput levels, we have created a genome-wide collection of human 3'UTR luciferase reporters in the highly-optimized LightSwitch Luciferase Assay System. The system also includes synthetic miRNA target reporter constructs for use as positive controls, various endogenous 3'UTR reporter constructs, and a series of standardized experimental protocols.
Here we describe a method for co-transfection of individual 3'UTR-reporter constructs along with a miRNA mimic that is efficient, reproducible, and amenable to high-throughput analysis.

MicroRNAs (miRNAs) are important regulators of gene expression and have been shown to play a role in numerous biological processes. To better understand miRNA-UTR interactions, we have created a genome-wide collection of 3 UTR luciferase reporters paired with a novel luciferase gene and assay reagent, the LightSwitch system.

模仿人类小分子RNA的LightSwitch萤光素酶检测系统使用3&#39;UTR记者构造的microRNA识别目标在贴壁细胞

MicroRNAs (miRNAs) are important regulators of gene expression and play a role in many biological processes.  More than 700 human miRNAs have been ...

Combining Optogenetics with Artificial microRNAs to Characterize the Effects of Gene Knockdown on Presynaptic Function within Intact Neuronal Circuits

The purpose of this protocol is to characterize the effect of gene knockdown on presynaptic function within intact neuronal circuits. We describe a workflow on how to combine artificial microRNA (miR)-mediated RNA interference with optogenetics to achieve selective stimulation of manipulated presynaptic boutons in acute brain slices. The experimental approach involves the use of a single viral construct and a single neuron-specific promoter to drive the expression of both an optogenetic probe and artificial miR(s) against presynaptic gene(s). When stereotactically injected in the brain region of interest, the expressed construct makes it possible to stimulate with light exclusively the neurons with reduced expression of the gene(s) under investigation. This strategy does not require the development and maintenance of genetically modified mouse lines and can in principle be applied to other organisms and to any neuronal gene of choice. We have recently applied it to investigate how the knockdown of alternative splice isoforms of presynaptic P/Q-type voltage-gated calcium channels (VGCCs) regulates short-term synaptic plasticity at CA3 to CA1 excitatory synapses in acute hippocampal slices. A similar approach could also be used to manipulate and probe the neuronal circuitry in vivo.

This protocol provides a workflow on how to combine artificial microRNA-mediated RNA interference with optogenetics to stimulate specifically presynaptic boutons with reduced expression of selective gene(s) within intact neuronal circuits.

光遗传学与人工 microRNAs 相结合对完整神经元回路中基因击倒对突触前功能的影响

The purpose of this protocol is to characterize the effect of gene knockdown on presynaptic function within intact neuronal circuits. We describe a ...

Loss-of-Function Approach in the Embryonic Chick Retina by Using Tol2 Transposon-Mediated Transgenic Expression of Artificial microRNAs

The chick retina has long been an important model system in developmental neurobiology, with advantages including its large size, rapid development, and accessibility for visualization and experimental manipulations. However, its major technical limitation had been the lack of robust loss-of-function approaches for gene function analyses. This protocol describes a methodology of gene silencing in the developing chick retina that involves transgenic expression of artificial microRNAs (miRNAs) by using the Tol2 transposon system. In this approach, a Tol2 transposon plasmid that contains an expression cassette for the EmGFP (emerald green fluorescent protein) marker and artificial pre-miRNA sequences against a target gene is introduced into the embryonic chick retina with a Tol2 transposase expression construct by in ovo electroporation. In the transfected retinal cells, the transposase catalyzes the excision of the expression cassette from the transposon vector and its integration into host chromosomes, leading to the stable expression of miRNAs and the EmGFP protein. In our previous study, we have demonstrated that the expression of Nel, a glycoprotein that exerts multiple functions in neural development, can be significantly suppressed in the developing chick retina by using this technique. Our results indicate that this methodology induces a stable and robust suppression of gene expression and thus provides an efficient loss-of-function approach for studies of retinal development.

We have developed a novel loss-of-function approach that involves the introduction and genomic integration of artificial micro-RNA sequences into chick embryos by using in ovo electroporation and the Tol2 transposon system. This technique provides a robust and stable gene knockdown methodology for studies of gene function during development.

使用Tol2转座子介导的人工microRNA的转基因表达在胚胎雏鸡视网膜中实现功能丧失的方法

The chick retina has long been an important model system in developmental neurobiology, with advantages including its large size, rapid development, and ...

一个简单的替代，以立体定向注射miRNA的脑特异性敲除

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此视频中的章节

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

基于生物素的下拉法对细胞 miRNAs mRNA 靶点的验证

胶质母细胞瘤中功能相关miRNA的特性及其工程在基因治疗的人工簇中

基于miRNA的质粒在发展中的神经管和表型的评估DiI荧光注射开卷准备在卵的电

设计吗啉反义寡核苷酸和 mRNA 并将其显微注射到斑马鱼胚胎中，以阐明心脏发育中的特定基因功能

设计吗啉反义寡核苷酸和 mRNA 并将其显微注射到斑马鱼胚胎中，以阐明心脏发育中的特定基因功能

miRNA的目标使用的任务目标ID库的全基因组屏幕

模仿人类小分子RNA的LightSwitch萤光素酶检测系统使用3'UTR记者构造的microRNA识别目标在贴壁细胞

光遗传学与人工 microRNAs 相结合对完整神经元回路中基因击倒对突触前功能的影响

使用Tol2转座子介导的人工microRNA的转基因表达在胚胎雏鸡视网膜中实现功能丧失的方法