这项工作是由国防部（理念发展奖，W81XWH-10-1-0041，AB），CT干细胞补助金（AB），和国立健康NRSA 10668225（DMF）的补助金。本发明的材料的基础工作部分由康涅狄格州的支持下，康涅狄格干细胞研究资助计划。它的内容是完全的责任作者的观点，并不一定代表国家的康涅狄格州，康涅狄格州或CT创新公共健康部的官方意见，公司的资助者不参与研究设计，数据收集分析，决定，公布，或准备的稿子。

本程序是在按照与耶鲁IACUC要求。科学家应确保IACUC准则的批准，随后根据其机构的要求。 1。第1节。制备DNA，解决方案和玻璃吸管<ol><li>生成高纯度（OD 260/280&gt; 1.80），高浓度（微克/微升&gt; 2.5）内毒素的DNA。 </li><li>准备0.9％的生理盐水溶液和无菌过滤器，通过一个22微米的过滤器。 </li><li>将10厘米火抛光的高硼硅玻璃毛细管（OD：1.5毫米，1.10毫米）到一个模型中PP-830成茂PC-10玻璃吸管车夫与康泰尔线。设置拉马一步加权拉在70.5°C。打破圆形钳的提示和检查锯齿状刀刃在解剖显微镜下。锥技巧和在紫外线灯下为15分钟。被拉移液器应标记的前端边缘在2毫米。 </li><li>准备0.1％重量/体积快速绿色的解决方案无菌过滤通过混合0.9％的生理盐水溶液用干燥的快绿。 </li></ol> 2。第2节。动物制备，玻璃吸管装载和质粒注射液<ol><li> 0-1幼崽分别从笼子里取出出生后，到玻璃培养皿预冷至4°C，然后湿冰的地方。 </li><li>大约5分钟后，确定麻醉状态的，利用脚捏响应。如果没有运动时，幼崽脑室注射。 </li><li>重要的是要注意，快速绿色，DNA，和盐溶液的组合可以导致的玻璃移液管的迅速蒸发，结晶，和封锁。因此，生成的储备液和等分试样1-2微升到石蜡紧接之前注射。 </li><li>拉玻璃吸管吸出整个等份量。 </li><li>保持你少惯用手的拇指和食指之间的小狗的头。 </li><li>灯的开启D保持头部的小狗，外面的光线。如果需要，用盐水头，以减少反射的光的量由小狗。心室现在应该亮起。 </li><li>你的惯用手，将针插入侧脑室最接近（ 图1A和1B）。注射的部位应是约从人字缝和眼睛和2毫米矢状缝外侧等距。拉移液管插入到2毫米大关的一个P0小狗，应保证焊透入侧脑室。巧合的是，你可能会看到回填土的CSF释放颅内压到吸管。为了降低颅内压，这有助于在注射质粒，放松你的抓地力头的pup.Step 2.8。约0.5μL的质粒注入侧脑室，使用高压空气喷射器（Picospritzer，帕克）。 </li></ol> 3。第3节。电穿孔和恢复<ol><li>硒t为5平方脉冲，50毫秒/脉冲在100伏，950毫秒的间隔电压电穿孔发生器。质粒电穿孔的空间特异性取决于由电荷转移的方向性。应给予密切关注的位置沿SVZ 8的干细胞群的异质性。已被确定为腹，背和头端尾部的位置9,10增殖和细胞的命运率的差异。 </li><li>一旦该质粒被注入到PBS，浸涂镊子电极。这一步是为了最大限度地提高电荷转移和防止烧伤引起的电阻率。 </li><li>放置的正电极上的背侧壁小狗质粒注射的部位（ 图1C和1D）同侧靠近耳朵。将负极放在对侧半球腹外侧的小狗的鼻子。 </li><li>按启动电流传输脉冲脚灯巫婆脚踏板和扫背的电极，横向〜25°角的时间间隔。 </li><li>广场电穿孔的幼崽到5分钟的加热垫。轻轻转动幼崽，每30秒有轻度刺激。 </li></ol>

<ol>
	<li>Kriegstein, A., Alvarez-Buylla, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+glial+nature+of+embryonic+and+adult+neural+stem+cells.">The glial nature of embryonic and adult neural stem cells.</a> Annual Review of Neuroscience. 32, 149-184 (2009).</li><li>Imayoshi, I., Sakamoto, M., Kageyama, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+methods+to+identify+and+manipulate+newly+born+neurons+in+the+adult+brain.">Genetic methods to identify and manipulate newly born neurons in the adult brain.</a> Frontiers in Neuroscience. 5, 64 (2011).</li><li>LoTurco, J., Manent, J. B., Sidiqi, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+and+improved+tools+for+in+utero+electroporation+studies+of+developing+cerebral+cortex.">New and improved tools for in utero electroporation studies of developing cerebral cortex.</a> Cereb Cortex. 19, i120-i125 (2009).</li><li>Tabata, H., Nakajima, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+in+utero+gene+transfer+system+to+the+developing+mouse+brain+using+electroporation:+visualization+of+neuronal+migration+in+the+developing+cortex.">Efficient in utero gene transfer system to the developing mouse brain using electroporation: visualization of neuronal migration in the developing cortex.</a> Neuroscience. 103, 865-872 (2001).</li><li>Boutin, C., Diestel, S., Desoeuvre, A., Tiveron, M. C., Cremer, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+in+vivo+electroporation+of+the+postnatal+rodent+forebrain.">Efficient in vivo electroporation of the postnatal rodent forebrain.</a> PloS ONE. 3, e1883 (2008).</li><li>Chesler, A. T., 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=Selective+gene+expression+by+postnatal+electroporation+during+olfactory+interneuron+nurogenesis.">Selective gene expression by postnatal electroporation during olfactory interneuron nurogenesis.</a> PloS ONE. 3, e1517 (2008).</li><li>Platel, J. C., 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=NMDA+receptors+activated+by+subventricular+zone+astrocytic+glutamate+are+critical+for+neuroblast+survival+prior+to+entering+a+synaptic+network.">NMDA receptors activated by subventricular zone astrocytic glutamate are critical for neuroblast survival prior to entering a synaptic network.</a> Neuron. 65, 859-872 (2010).</li><li>Alvarez-Buylla, A., Kohwi, M., Nguyen, T. M., Merkle, F. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+heterogeneity+of+adult+neural+stem+cells+and+the+emerging+complexity+of+their+niche.">The heterogeneity of adult neural stem cells and the emerging complexity of their niche.</a> Cold Spring Harbor Symposia on Quantitative Biology. 73, 357-365 (2008).</li><li>Fernandez, M. E., Croce, S., Boutin, C., Cremer, H., Raineteau, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeted+electroporation+of+defined+lateral+ventricular+walls:+a+novel+and+rapid+method+to+study+fate+specification+during+postnatal+forebrain+neurogenesis.">Targeted electroporation of defined lateral ventricular walls: a novel and rapid method to study fate specification during postnatal forebrain neurogenesis.</a> Neural Development. 6, 13 (2011).</li><li>de Chevigny, A., 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=miR-7a+regulation+of+Pax6+controls+spatial+origin+of+forebrain+dopaminergic+neurons.">miR-7a regulation of Pax6 controls spatial origin of forebrain dopaminergic neurons.</a> Nature Neuroscience. 15, 1120-1126 (2012).</li><li>Lacar, B., Young, S. Z., Platel, J. C., Bordey, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+and+recording+subventricular+zone+progenitor+cells+in+live+tissue+of+postnatal+mice.">Imaging and recording subventricular zone progenitor cells in live tissue of postnatal mice.</a> Frontiers in Neuroscience. 4, (2010).</li><li>Feliciano, D. M., Quon, J. L., Su, T., Taylor, M. M., Bordey, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Postnatal+neurogenesis+generates+heterotopias,+olfactory+micronodules+and+cortical+infiltration+following+single-cell+Tsc1+deletion.">Postnatal neurogenesis generates heterotopias, olfactory micronodules and cortical infiltration following single-cell Tsc1 deletion.</a> Human Molecular Genetics. 21, 799-810 (2012).</li><li>Iguchi, T., Yagi, H., Wang, C. C., Sato, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+tightly+controlled+conditional+knockdown+system+using+the+Tol2+transposon-mediated+technique.">A tightly controlled conditional knockdown system using the Tol2 transposon-mediated technique.</a> PloS ONE. 7, e33380 (2012).</li></ol>

作者没有披露。

在这里，我们详细新生儿SVZ电的技术，这种技术能够迅速和有力的标记和操作SVZ干细胞和它们的后代。有几个优点，在与其它技术相比，电穿孔具有。首先，考虑的重点标记的细胞，一个是能分辨细胞自主和非细胞自主的影响。其次，使用诱导系统的遗传操作，允许一个用来比较的效果之前或之后突触融合。此外，人们可以绕过使用多个质粒与含有loxP位点的质粒通过电穿孔CRE表达小鼠。第四，报...

<table border="1"><tbody><tr><td> 的试剂的名称 </td><td> 公司 </td><td> 目录编号 </td><td> 评论（可选） </td></tr><tr><td>重抛光的高硼硅管</td><td>萨特仪器</td><td> BF150-110-10 </td><td></td></tr><tr><td>双级玻璃微普勒</td><td>成重</td><td> PC-10H </td><td></td></tr><tr><td>固绿</td><td>菲舍尔科学</td><td> 0521192205 </td><td></td></tr><tr><td> ECM 830方波脉冲发生器</td><td>哈佛设备</td><td> 45-0052 </td><td></td></tr><tr><td> Tweezertrodes </td><td>哈佛设备</td><td> 45-0488 </td><td></td></tr><tr><td>光纤光源</td><td> Fisher Scientific则</td><td> 12-562-36 </td><td></td></tr><tr><td>卤钨灯灯</td><td> USHIO美国公司</td><td> 1002247 </td><td></td></tr><tr><td> Picospritzer II </td><td>帕克工具</td><td> 052-0312-900 </td><td></td></tr></tbody></table>

neonatal subventricular zone electroporation

神经干细胞（NSCs）线的产后侧脑室，并产生多种细胞类型，包括神经元，星形胶质细胞和室管膜细胞。了解，利用其独特的修复潜力更好地理解大脑和中枢神经系统紊乱的分子通路的负责NSC的自我更新，承诺和分化是至关重要的。往前哺乳动物系统操纵的方法需要耗费时间和昂 ​​贵的基因工程的努力在整个动物的第2级。因此，绝大多数研究探讨NSC分子在体外或在无脊椎动物中的功能。 在这里，我们展示了技术操作简单，快速的新生儿的NPC，被称为新生儿脑室下区（SVZ）电。十年前制定类似的技术来研究胚胎神经干细胞，并资助研究cortical开发3,4。最近研究产后的啮齿动物前脑5-7。这种技术的结果SVZ神经干细胞在强大的标签和他们的后代。因此，产后SVZ电哺乳动物国科会基因工程提供了成本和时间效益的替代方案。

新生儿脑室下区电在几乎所有的放射状胶质连续的背，背外侧和侧脑室下区继“清扫”运动的镊子电极（ 图1E）的标签。然而，电可定制的要求，在各自的实验，但不扫电极，并使用特定的位置和方向，详细的视频。例如，由于背侧局部放射状胶质不同于那些衬心室的横向部分，一个可以选择只有一个单一的区域9,10 electroporate。更多特殊性的也可以使用较小的电极。 <p clas...

Watch this Scientific Journal Video about Neonatal Subventricular Zone Electroporation at JoVE.com

Neonatal Subventricular Zone Electroporation

我们展示出一种微创技术，新生儿脑室下区电。该技术包括新生幼崽的侧脑室注射质粒DNA和通电时提供神经干细胞和基因操纵

新生儿脑室下区电穿孔

Neural stem cells (NSCs) line the postnatal lateral ventricles and give rise to multiple cell types which include neurons, astrocytes, and ependymal ...

neonatal-subventricular-zone-electroporation

Research

JoVE Journal

Neuroscience

12.7K 次观看.  Yale University School of Medicine. 我们展示出一种微创技术，新生儿脑室下区电。该技术包括新生幼崽的侧脑室注射质粒DNA和通电时提供神经干细胞和基因操纵

视频: Neonatal Subventricular Zone Electroporation

The Subventricular Zone En-face: Wholemount Staining and Ependymal Flow

The walls of the lateral ventricles contain the largest germinal region in the adult mammalian brain. The subventricular zone (SVZ) in these walls is an extensively studied model system for understanding the behavior of neural stem cells and the regulation of adult neurogenesis. Traditionally, these studies have relied on classical sectioning techniques for histological analysis. Here we present an alternative approach, the wholemount technique, which provides a comprehensive, en-face view of this germinal region. Compared to sections, wholemounts preserve the complete cytoarchitecture and cellular relationships within the SVZ. This approach has recently revealed that the adult neural stem cells, or type B1 cells, are part of a mixed neuroepithelium with differentiated ependymal cells lining the lateral ventricles. In addition, this approach has been used to study the planar polarization of ependymal cells and the cerebrospinal fluid flow they generate in the ventricle. With recent evidence that adult neural stem cells are a heterogeneous population that is regionally specified, the wholemount approach will likely be an essential tool for understanding the organization and parcellation of this stem cell niche.

The lateral ventricle walls contain the largest germinal region in the adult mammalian brain. Traditionally, studies on neurogenesis in this region have relied on classical sectioning techniques for histological analysis. Here we present an alternative approach, the wholemount technique, which provides a comprehensive, en-face view of this germinal region.

脑室下区恩的脸：Wholemount染色和室管膜流量

The walls of the lateral ventricles contain the largest germinal region in the adult mammalian brain. The subventricular zone (SVZ) in these walls is an ...

科研

神经科学

In vivo Electroporation of Developing Mouse Retina

The functional characterization of genes expressed during mammalian retinal development remains a significant challenge. Gene targeting to generate constitutive or conditional loss of function knockouts remains cost and labor intensive, as well as time consuming. Adding to these challenges, retina expressed genes may have essential roles outside the retina leading to unintended confounds when using a knockout approach. Furthermore, the ability to ectopically express a gene in a gain of function experiment can be extremely valuable when attempting to identify a role in cell fate specification and/or terminal differentiation.
We present a method for the rapid and efficient incorporation of DNA plasmids into the neonatal mouse retina by electroporation. The application of short electrical impulses above a certain field strength results in a transient increase in plasma membrane permeability, facilitating the transfer of material across the membrane 1,2,3,4. Groundbreaking work demonstrated that electroporation could be utilized as a method of gene transfer into mammalian cells by inducing the formation of hydrophilic plasma membrane pores allowing the passage of highly charged DNA through the lipid bilayer 5. Continuous technical development has resulted in the viability of electroporation as a method for in vivo gene transfer in multiple mouse tissues including the retina, the method for which is described herein 6, 7, 8, 9, 10. 
DNA solution is injected into the subretinal space so that DNA is placed between the retinal pigmented epithelium and retina of the neonatal (P0) mouse and electrical pulses are applied using a tweezer electrode. The lateral placement of the eyes in the mouse allows for the easy orientation of the tweezer electrode to the necessary negative pole-DNA-retina-positive pole alignment. Extensive incorporation and expression of transferred genes can be identified by postnatal day 2 (P2). Due to the lack of significant lateral migration of cells in the retina, electroporated and non-electroporated regions are generated. Non-electroporated regions may serve as internal histological controls where appropriate. 
Retinal electroporation can be used to express a gene under a ubiquitous promoter, such as CAG, or to disrupt gene function using shRNA constructs or Cre-recombinase. More targeted expression can be achieved by designing constructs with cell specific gene promoters. Visualization of electroporated cells is achieved using bicistronic constructs expressing GFP or by co-electroporating a GFP expression construct. Furthermore, multiple constructs may be electroporated for the study of combinatorial gene effects or simultaneous gain and loss of function of different genes. Retinal electroporation may also be utilized for the analysis of genomic cis-regulatory elements by generating appropriate expression constructs and deletion mutants. Such experiments can be used to identify cis-regulatory regions sufficient or required for cell specific gene expression 11. Potential experiments are limited only by construct availability.

In vivo Electroporation of Developing Mouse Retina

A method for the incorporation of plasmid DNA into murine retinal cells for the purpose of performing either gain- or loss of function studies in vivo is presented. This method capitalizes on the transient increase in permeability of cell plasma membranes induced by the application of an external electrical field.

在发展中国家小鼠视网膜体内电穿孔

The functional characterization of genes expressed during mammalian retinal development remains a significant challenge.  Gene targeting to generate ...

Preparation of Acute Subventricular Zone Slices for Calcium Imaging

The subventricular zone (SVZ) is one of the two neurogenic zones in the postnatal brain. The SVZ contains densely packed cells, including neural progenitor cells with astrocytic features (called SVZ astrocytes), neuroblasts, and intermediate progenitor cells. Neuroblasts born in the SVZ tangentially migrate a great distance to the olfactory bulb, where they differentiate into interneurons. Intercellular signaling through adhesion molecules and diffusible signals play important roles in controlling neurogenesis. Many of these signals trigger intercellular calcium activity that transmits information inside and between cells. Calcium activity is thus reflective of the activity of extracellular signals and is an optimal way to understand functional intercellular signaling among SVZ cells.
Calcium activity has been studied in many other regions and cell types, including mature astrocytes and neurons. However, the traditional method to load cells with calcium indicator dye (i.e. bath loading) was not efficient at loading all SVZ cell types. Indeed, the cellular density in the SVZ precludes dye diffusion inside the tissue. In addition, preparing sagittal slices will better preserve the three-dimensional arrangement of SVZ cells, particularly the stream of neuroblast migration on the rostral-caudal axis.
Here, we describe methods to prepare sagittal sections containing the SVZ, the loading of SVZ cells with calcium indicator dye, and the acquisition of calcium activity with time-lapse movies. We used Fluo-4 AM dye for loading SVZ astrocytes using pressure application inside the tissue. Calcium activity was recorded using a scanning confocal microscope allowing a precise resolution for distinguishing individual cells. Our approach is applicable to other neurogenic zones including the adult hippocampal subgranular zone and embryonic neurogenic zones. In addition, other types of dyes can be applied using the described method.

A method to load subventricular zone (SVZ) cells with calcium indicator dyes for recording calcium activity is described. The postnatal SVZ contains tightly packed cells including neural progenitor cells and neuroblasts. Rather than using bath loading we injected the dye by pressure inside the tissue allowing better dye diffusion.

钙成像的急性下区片的制备

The subventricular zone (SVZ) is one of the two neurogenic zones in the postnatal brain. The SVZ contains densely packed cells, including neural ...

Neonatal Pial Surface Electroporation

Over the past several years the pial surface has been identified as a germinal niche of importance during embryonic, perinatal and adult neuro- and gliogenesis, including after injury. However, methods for genetically interrogating these progenitor populations and tracking their lineages had been limited owing to a lack of specificity or time consuming production of viruses. Thus, progress in this region has been relatively slow with only a handful of investigations of this location. Electroporation has been used for over a decade to study neural stem cell properties in the embryo, and more recently in the postnatal brain. Here we describe an efficient, rapid, and simple technique for the genetic manipulation of pial surface progenitors based on an adapted electroporation approach. Pial surface electroporation allows for facile genetic labeling and manipulation of these progenitors, thus representing a time-saving and economical approach for studying these cells.

The pial surface is a unique progenitor zone in the CNS that is receiving increasing attention. Herein, we detail a method for rapid genetic manipulation of this progenitor zone using a modified electroporation method. This procedure can be used for cellular and molecular investigations of cell lineages and signaling pathways involved in cell differentiation and to elucidate the fate and properties of daughter cells.

新生儿软脑膜表面电穿孔

Over the past several years the pial surface has been identified as a germinal niche of importance during embryonic, perinatal and adult neuro- and ...

Subtype-selective Electroporation of Cortical Interneurons

The study of central nervous system (CNS) maturation relies on genetic targeting of neuronal populations. However, the task of restricting the expression of genes of interest to specific neuronal subtypes has proven remarkably challenging due to the relative scarcity of specific promoter elements. GABAergic interneurons constitute a neuronal population with extensive genetic and morphological diversity. Indeed, more than 11 different subtypes of GABAergic interneurons have been characterized in the mouse cortex1. Here we present an adapted protocol for selective targeting of GABAergic populations. We achieved subtype selective targeting of GABAergic interneurons by using the enhancer element of the homeobox transcription factors Dlx5 and Dlx6, homologues of the Drosophila distal-less (Dll) gene2,3, to drive the expression of specific genes through in utero electroporation.

This procedure shows how to target interneurons in the developing mouse forebrain by means of in utero electroporation. This technique was particularly efficient to achieve selective gene expression in interneuron subtypes destined to the superficial layers of the cortex.

皮质的interneurons的亚型选择性电穿孔

The study of central nervous system (CNS) maturation relies on genetic targeting of neuronal populations. However, the task of restricting the expression ...

Cell Sorting of Neural Stem and Progenitor Cells from the Adult Mouse Subventricular Zone and Live-imaging of their Cell Cycle Dynamics

Neural stem cells (NSCs) in the subventricular zone of the lateral ventricles (SVZ) sustain olfactory neurogenesis throughout life in the mammalian brain. They successively generate transit amplifying cells (TACs) and neuroblasts that differentiate into neurons once they integrate the olfactory bulbs. Emerging fluorescent activated cell sorting (FACS) techniques have allowed the isolation of NSCs as well as their progeny and have started to shed light on gene regulatory networks in adult neurogenic niches. We report here a cell sorting technique that allows to follow and distinguish the cell cycle dynamics of the above-mentioned cell populations from the adult SVZ with a LeX/EGFR/CD24 triple staining. Isolated cells are then plated as adherent cells to explore in details their cell cycle progression by time-lapse video microscopy. To this end, we use transgenic Fluorescence Ubiquitination Cell Cycle Indicator (FUCCI) mice in which cells are red-fluorescent during G1 phase due to a G1 specific red-Cdt1 reporter. This method has recently revealed that proliferating NSCs progressively lengthen their G1 phase during aging, leading to neurogenesis impairment. This method is easily transposable to other systems and could be of great interest for the study of the cell cycle dynamics of brain cells in the context of brain pathologies.

We report a Fluorescent Activated Cell Sorting (FACS)-based method to isolate neural stem cells (NSCs) and their progeny from the subventricular zone (SVZ) of the adult mouse brain. Applied to Fluorescence Ubiquitination Cell Cycle Indicator (FUCCI) transgenic mice, it allows the study of cell cycle progression by live imaging.

神经干细胞和祖细胞来自成年小鼠脑室下区细胞分选和实时成像的细胞周期动力学

Neural stem cells (NSCs) in the subventricular zone of the lateral ventricles (SVZ) sustain olfactory neurogenesis throughout life in the mammalian brain. ...

Isolation and Culture of Adult Neural Stem Cells from the Mouse Subcallosal Zone

Adult neural stem cells (aNSCs) can be used for the regeneration of damaged brain tissue. NSCs have the potential for differentiation and proliferation into three types of cells: neurons, astrocytes, and oligodendrocytes. Identifying aNSC-derived regions and characterizing the aNSC properties are critical for the potential use of aNSCs and for the elucidation of their role in neural regeneration. The subcallosal zone (SCZ), located between white matter and the hippocampus, has recently been reported to contain aNSCs and continuously give rise to neuroblasts. A low percentage of aNSCs from the SCZ is differentiated into neurons; most cells are differentiated into glial cells, such as oligodendrocytes and astrocytes. These cells are suggested to have a therapeutic potential for traumatic cortical injury. This protocol describes in detail the process to generate SCZ-aNSCs from an adult mouse brain. A brain matrix with intervals of 1 mm is used to obtain the SCZ-containing coronal slices and to precisely dissect the SCZ from the whole brain. The SCZ sections are initially subjected to a neurosphere culture. A well-developed culture system allows for the verification of their characteristics and can increase research on NSCs. A neurosphere culture system provides a useful tool for determining proliferation and collecting the genuine NSCs. A monolayer culture is also an in vitro system to assay proliferation and differentiation. Significantly, this culture system provides a more homogenous environment for NSCs than the neurosphere culture system. Thus, using a discrete brain region, these culture systems will be helpful for expanding our knowledge about aNSCs and their applications for therapeutic uses.

Establishing culture systems for the expansion of adult neural stem cells (aNSCs) allows for the examination and application of aNSCs for therapy. The subcallosal zone (SCZ) has recently been recognized as a novel neuroblast-forming region in adult mice. Here, methods for the isolation, expansion, and differentiation of SCZ-aNSCs are described.

分离和成人神经干细胞的培养从鼠标Subcallosal区

Adult neural stem cells (aNSCs) can be used for the regeneration of damaged brain tissue. NSCs have the potential for differentiation and proliferation ...

Cryo-section Dissection of the Adult Subependymal Zone for Accurate and Deep Quantitative Proteome Analysis

The subependymal neurogenic niche consists of a paraventricular ribbon of the lateral ventricular wall of the lateral ventricle. The subependymal zone (SEZ) is a thin and distinct region exposed to the ventricles and cerebrospinal fluid. The isolation of this niche allows the analysis of a neurogenic stem cell microenvironment. However, extraction of small tissues for proteome analysis&#160;is challenging, especially for the maintenance of considerable measurement depth and the achievement of reliable robustness. A new method termed cryo-section-dissection (CSD), combining high precision with minimal tissue perturbation, was developed to address these challenges. The method is compatible with state-of-the-art mass spectrometry (MS) methods that allow the detection of low-abundant niche regulators. This study compared the CSD and its proteome data to the method and data obtained by laser-capture-microdissection (LCM) and a standard wholemount dissection. The CSD method resulted in twice the quantification depth in less than half the preparation time compared to the LCM and simultaneously clearly outperformed the dissection precision of the wholemount dissection. Hence, CSD is a superior method for collecting the SEZ for proteome analysis.

Cryo-section-dissection allows fresh, frozen preparation of the largest neurogenic niche in the murine brain for deep quantitative proteome analysis. The method is precise, efficient, and causes minimal tissue perturbation. Therefore, it is ideally suited for studying the molecular microenvironment of this niche, as well as other organs, regions, and species.

冷冻切片解剖成人室管膜下区，用于准确和深入的定量蛋白质组分析

The subependymal neurogenic niche consists of a paraventricular ribbon of the lateral ventricular wall of the lateral ventricle. The subependymal zone ...

A Neonatal Rodent Model of Retroorbital Vein Injection

Intravenous (iv) injection is the most used route of drug administration in neonates in the clinical setting. Therefore, retroorbital vein injection is an important method for compound administration in research, where successful proof-of-concept studies can progress into much-needed neonatal clinical trials. Most intravenous studies in neonatal rodents use the superficial temporal/facial vein. However, retroorbital injection becomes unreliable in neonatal rodents older than 2 days after the skin darkens and the vein is no longer visible. In the present protocol, we describe the retroorbital injection of the venous sinus in both the neonatal mouse and rat at ages when the superficial temporal vein is no longer visible, but the eyes have not opened yet. Eye-opening facilitates retro-orbital injection by enabling the researcher to clearly see that they are not perforating the eye when inserting the needle. We demonstrate that this technique can be performed in a reliable and reproducible manner without adverse effects. Additionally, we show that it can be used in many studies, such as administering compounds to study neonatal brain injury.

This protocol aims to demonstrate a reproducible venous administration route that can be used in rats and mice throughout the neonatal period. This procedure is important for preclinical rodent studies that wish to mirror drug administration in neonatal care units primarily using intravenous administration.

眶后静脉注射的新生儿啮齿动物模型

Intravenous (iv) injection is the most used route of drug administration in neonates in the clinical setting. Therefore, retroorbital vein injection is an ...

<meta charset="utf8"></head><body>对从小鼠脑室下区分离的 NSC 进行高效成核

Isolation, Expansion, and Nucleofection of Neural Stem Cells from Adult Murine Subventricular Zone

Isolation and expansion of neural stem cells (NSCs) from the subventricular zone (SVZ) of the adult mouse brain can be achieved in a medium supplemented with basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF) as mitogens, producing clonal aggregates known as neurospheres. This in vitro system is a valuable tool for studying NSC potential. Transfection of siRNAs or genes carried in plasmids can be used to induce perturbations to gene expression and study NSC biology. However, the exogenous nucleic acid delivery to NSC cultures is challenging due to the low efficiency of central nervous system (CNS) cells transfection. Here, we present an improved nucleofection system that achieves high efficiency of gene delivery in expanded NSCs from adult murine SVZ. We demonstrate that this relatively simple method enhances gene perturbation in adult NSCs, surpassing traditional transfection protocols with survival rates exceeding 80%. Moreover, this method can also be applied in primary isolated NSCs, providing a crucial advancement in gene function studies through gene expression manipulation via knockdown or overexpression in neurosphere cultures.

<meta charset="utf8"></head><body>作者聚焦：改进的核转染技术，在小鼠脑室下区衍生的神经干细胞培养物中实现高效基因递送

Here, we describe a nucleofection system designed to enhance gene delivery efficiency in expanded neural stem cells (NSCs) isolated from the adult murine subventricular zone. The findings demonstrate that this method significantly improves gene perturbation in NSCs, surpassing the effectiveness of traditional transfection protocols and enhancing cell survival rate.

来自成体小鼠脑室下区的神经干细胞的分离、扩增和成核转染

Isolation and expansion of neural stem cells (NSCs) from the subventricular zone (SVZ) of the adult mouse brain can be achieved in a medium supplemented ...