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>

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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

Systemic Delivery of MicroRNA Using Recombinant Adeno-associated Virus Serotype 9 to Treat Neuromuscular Diseases in Rodents

RNA interference via the endogenous miRNA pathway regulates gene expression by controlling protein synthesis through post-transcriptional gene silencing. In recent years, miRNA-mediated gene regulation has shown potential for treatment of neurological disorders caused by a toxic gain of function mechanism. However, efficient delivery to target tissues has limited its application. Here we used a transgenic mouse model for spinal and bulbar muscular atrophy (SBMA), a neuromuscular disease caused by polyglutamine expansion in the androgen receptor (AR), to test gene silencing by a newly identified AR-targeting miRNA, miR-298. We overexpressed miR-298 using a recombinant adeno-associated virus (rAAV) serotype 9 vector to facilitate transduction of non-dividing cells. A single tail-vein injection in SBMA mice induced sustained and widespread overexpression of miR-298 in skeletal muscle and motor neurons and resulted in amelioration of the neuromuscular phenotype in the mice.

Here we describe the delivery of microRNA using a recombinant adeno-associated virus serotype 9 in a mouse model of a neuromuscular disease. A single peripheral administration in mice resulted in sustained miRNA overexpression in muscle and motor neurons, providing an opportunity to study miRNA function and therapeutic potential in vivo.

重组腺相关病毒9型 MicroRNA 系统分娩治疗啮齿动物神经肌肉疾病

RNA interference via the endogenous miRNA pathway regulates gene expression by controlling protein synthesis through post-transcriptional gene silencing. ...

遗传学

In Vivo Inhibition of MicroRNA to Decrease Tumor Growth in Mice

MicroRNAs (miRNAs) are important regulators of gene expression through their ability to destabilize mRNA and inhibit translation of target mRNAs. An ever-increasing number of studies have identified miRNAs as potential biomarkers for cancer diagnosis and prognosis, and also as therapeutic targets, adding an extra dimension to cancer evaluation and treatment. In the context of thyroid cancer, tumorigenesis results not only from mutations in important genes, but also from the overexpression of many miRNAs. Accordingly, the role of miRNAs in the control of thyroid gene expression is evolving as an important mechanism in cancer. Herein, we present a protocol to examine the effects of miRNA-inhibitor delivery as a therapeutic modality in thyroid cancer using human tumor xenograft and orthotopic mouse models. After engineering stable thyroid tumoral cells expressing GFP and luciferase, cells are injected into nude mice to develop tumors, which can be followed by bioluminescence. The in vivo inhibition of a miRNA can reduce tumor growth and upregulate miRNA gene targets. This method can be used to assess the importance of a determined miRNA in vivo, in addition to identifying new therapeutic targets.

This protocol describes xenograft and orthotopic mouse models of human thyroid tumorigenesis as a platform to test microRNA-based inhibitor treatments. This approach is ideal to study the function of non-coding RNAs and their potential as new therapeutic targets.

在微RNA的Vivo抑制下，降低小鼠的肿瘤生长

MicroRNAs (miRNAs) are important regulators of gene expression through their ability to destabilize mRNA and inhibit translation of target mRNAs. An ...

癌症研究

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

A Positioning Device for the Placement of Mice During Intranasal siRNA Delivery to the Central Nervous System

Intranasal (IN) drug delivery to the brain has emerged as a promising method to bypass the blood-brain barrier (BBB) for the delivery of drugs into the central nervous system (CNS). Recent studies demonstrate the use of a peptide, RVG9R, incorporating the minimal receptor-binding domain of the Rabies virus glycoprotein, in eliciting the delivery of siRNA into neurons in the brain. In this protocol, the peptide-siRNA formulation is delivered intranasally with a pipette in the dominant hand, while the anesthetized mouse is restrained by the scruff with the nondominant hand in a "head down-and-forward position" to avoid drainage into the lung and stomach upon inhalation. This precise gripping of mice can be learned but is not easy and requires practice and skill to result in effective CNS uptake. Furthermore, the process is long-drawn, requiring about 45 min for the administration of a total volume of ~20-30 &#181;L of solution in 1-2 &#181;L droplet volumes per inhalation, with 3-4 min rest periods between each inhalation. The objective of this study is to disclose a mouse positioning device that enables the appropriate placement of mice for efficient IN administration of the peptide-siRNA formulation. Multiple features are incorporated into the design of the device, such as four or eight positioning chairs with adjustable height and tilt to restrain anesthetized mice in the head down-and-forward position, enabling easy visualization of the mice's nares and a built-in heating pad to maintain the mice's body temperatures during the procedure. Importantly, the ability to treat four or eight mice simultaneously with RVG9R-siRNA complexes in this manner enables studies on a much quicker time scale, for the testing of an IN therapeutic siRNA approach. In conclusion, this device allows for appropriate and controlled mouse head positioning for IN application of RVG9R-siRNA and other therapeutic molecules, such as nanoparticles or antibodies, for CNS delivery.

Here, we present a protocol using a mouse positioning device that enables the appropriate placement of mice for the intranasal administration of a brain-targeting peptide-siRNA formulation enabling effective gene silencing in the central nervous system.

在鼻内siRNA输送到中枢神经系统期间放置小鼠的定位装置

Intranasal (IN) drug delivery to the brain has emerged as a promising method to bypass the blood-brain barrier (BBB) for the delivery of drugs into the ...

免疫与感染

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 ...

In Vivo Nanovector Delivery of a Heart-specific MicroRNA-sponge

MicroRNA (miRNA) is small non-coding RNA which inhibits post-transcriptional messenger RNA (mRNA) expression. Human diseases, such as cancer and cardiovascular disease, have been shown to activate tissue and/or cell-specific miRNA expression associated with disease progression. The inhibition of miRNA expression offers the potential for a therapeutic intervention. However, traditional approaches to inhibit miRNAs, employing antagomir oligonucleotides, affect specific miRNA functions upon global delivery. Herein, we present a protocol for the in vivo cardio-specific inhibition of the miR-181 family in a rat model. A miRNA-sponge construct is designed to include 10 repeated anti-miR-181 binding sequences. The cardio-specific &#945;-MHC promoter is cloned into the pEGFP backbone to drive the cardio-specific miR-181 miRNA-sponge expression. To create a stable cell line expressing the miR-181-sponge, myoblast H9c2 cells are transfected with the &#945;-MHC-EGFP-miR-181-sponge construct and sorted by fluorescence-activated cell sorting (FACs) into GFP positive H9c2 cells which are cultured with neomycin (G418). Following stable growth in neomycin, monoclonal cell populations are established by additional FACs and single cell cloning. The resulting myoblast H9c2-miR-181-sponge-GFP cells exhibit a loss of function of miR-181 family members as assessed through the increased expression of miR-181 target proteins and compared to H9c2 cells expressing a scramble non-functional sponge. In addition, we develop a nanovector for the systemic delivery of the miR-181-sponge construct by complexing positively charged liposomal nanoparticles and negatively charged miR-181-sponge plasmids. In vivo imaging of GFP reveals that multiple tail vein injections of a nanovector over a three-week period are able to promote a significant expression of the miR-181-sponge in a cardio-specific manner. Importantly, a loss of miR-181 function is observed in the heart tissue but not in the kidney or the liver. The miRNA-sponge is a powerful method to inhibit tissue-specific miRNA expression. Driving the miRNA-sponge expression from a tissue-specific promoter provides specificity for the miRNA inhibition, which can be confined to a targeted organ or tissue. Furthermore, combining nanovector and miRNA-sponge technologies permits an effective delivery and tissue-specific miRNA inhibition in vivo.

In Vivo Nanovector Delivery of a Heart-specific MicroRNA-sponge

Tissue-specific microRNA inhibition is a technology that is underdeveloped in the microRNA field. Herein, we describe a protocol to successfully inhibit the miR-181 microRNA family in myoblast cells from the heart. Nanovector technology is used to deliver a microRNA sponge that demonstrates significant in vivo cardio-specific miR-181 family inhibition.

体内Nanovector 心脏特异性 MicroRNA 海绵的交付

MicroRNA (miRNA) is small non-coding RNA which inhibits post-transcriptional messenger RNA (mRNA) expression. Human diseases, such as cancer and ...

生物学

CRISPR Gene Editing Tool for MicroRNA Cluster Network Analysis

MicroRNAs (miRNAs) have emerged as important cellular regulators (tumor suppressors, pro-oncogenic factors) of cancer and metastasis. Most published studies focus on a single miRNA when characterizing the role of small RNAs in cancer. However, ~30%&#160;of human miRNA genes are organized in clustered units that are often co-expressed, indicating a complex and coordinated system of noncoding RNA regulation. A clearer understating of how clustered miRNA networks function cooperatively to regulate tumor growth, cancer aggressiveness, and drug resistance is required before translating noncoding small RNAs to the clinic.
The use of a high-throughput clustered regularly interspaced short palindromic repeats (CRISPR)-mediated gene editing procedure has been employed to study the oncogenic role of a genomic cluster of seven miRNA genes located within a locus spanning ~35,000 bp in length in the context of prostate cancer. For this approach, human cancer cell lines were infected with a lentivirus vector for doxycycline (DOX)-inducible Cas9 nuclease grown in DOX-containing medium for 48 h. The cells were subsequently co-transfected with synthetic trans-activating CRISPR RNA (tracrRNA) complexed with genomic site-specific CRISPR RNA (crRNA) oligonucleotides to allow the rapid generation of cancer cell lines carrying the entire miRNA cluster deletion and individual or combination miRNA gene cluster deletions within a single experiment.
The advantages of this high-throughput gene editing system are the ability to avoid time-consuming DNA vector subcloning, the flexibility in transfecting cells with unique guide RNA combinations in a 24-well format, and the lower-cost PCR genotyping using crude cell lysates. Studies using this streamlined approach promise to uncover functional redundancies and synergistic/antagonistic interactions between miRNA cluster members, which will aid in characterizing the complex small noncoding RNA networks involved in human disease and better inform future therapeutic design.

This protocol describes a high-throughput clustered regularly interspaced short palindromic repeats (CRISPR) gene editing workflow for microRNA cluster network analysis that allows the rapid generation of a panel of genetically modified cell lines carrying unique miRNA cluster member deletion combinations as large as 35 kb within a single experiment.

用于微RNA簇网络分析的CRISPR基因编辑工具

MicroRNAs (miRNAs) have emerged as important cellular regulators (tumor suppressors, pro-oncogenic factors) of cancer and metastasis. Most published ...