NIH 그랜트 HD - 32116, 재단, 존 보스 줄기 세포 기금, MEXT, MHLW 및 HFSP를 지원하는 샌들러 가족 지원 작업. ZM는 카를로스 Baldoceda 재단과 UCSF Krevans 원정대에 의해 지원. AA - B. 헤더와 멜라니는 둘러 싸인 의자를 혼란 보유하고 있습니다.

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심실과 subventricular 영역에서 neurogenesis 대부분의 연구 microanatomy 이러한 지역에서 휴대 관계를 조사하는 클래식 sectioning 기술에 의존합니다. 여기 우리는 다음 방지 mitotic 처리 6, 그리고 가장 최근에 정확한 혀끝의 연구를하는 데 사용되는 다음과 같은 SVZ의 시조 인구의 재생을 연구하는 데 먼저 SVZ 1 생성 neuroblasts의 철새 체인의 네트워크를 분석하는 데 사용되는 대체 기술, 설명 성인 SVZ 신경 줄기 세포 2,3,4의 기저 세포 - 세포 상호 작용. 흥미롭게도,이 기술은 성인 SVZ의 신경 줄기 세포 또는 형식 B1 세포가 차별이 아닌 나누어 ependymal 전지와 혼합 neuroepithelium의 일부입니다 것으로 나타났습니다. wholemounts를 사용 엉 - 얼굴 영상이 혼합 neuroepithelium는 ependymal 세포 4 대형 혀끝의 표면에 둘러싸여 타입 B1 세포의 꼭대기의 엔딩 구성된 바람개비 아키텍처를 가지고 나타났습니다. 이 엉 - 얼굴 분석 혈관 틈새 문의 뇌실 표면에 혀끝의 엔딩과 기초 프로세스 세포의 구성으로 배아와 성인 두뇌의 신경 줄기 세포의 혈통에 대한 우리의 이해를 명확히하고 있습니다. 이러한 연구 결과는 고전 sectioning 기술을 사용하여 거의 불가능했을. Wholemounts들은 또한 뇌실 - 문의 혀끝의 과정을 통해 신경 줄기 세포의 식별을 용이하게합니다. 이러한 줄기 세포에 대한 더 구체적인 마커가 발견됨에 wholemounts는 신경 줄기 세포의 행동을 식별하고 분석의 중요한 부분이 될 것입니다. 측면 뇌실 벽의 Wholemounts도 ependymal 세포의 평면 극성을 공부하고 이상적인 관점을 제공합니다. Ependymal 세포는 조정 방식으로 CSF를 추진하기 위해 함수 심실을 안감 multiciliated 세포 수 있습니다. wholemount 기술과 함께, 전체 ependymal 상피는 엉 - 얼굴을 노출하고 사후하고 복부 등의 경계에 스테인드하고 앞쪽에에서 종합적으로 연구하실 수 있습니다. 또한, ependymal 흐름 assays가 심하게 해부에 수행, 라이브 wholemounts는 견고 ependymal 속눈썹에 의해 생성되는 평면 편광 흐름을 보여줍니다. wholemount 접근법을 사용하는 최근의 작품이 ependymal 평면 극성 5 determinants 세포를 발견했다. 흥미롭게도, wholemount 연구는 ependymal 생성된 CSF의 흐름 SVZ 7 젊은 뉴런의 마이 그 레이션 가이드 chemorepellents의 그라디언트를 설립 것을 제안했습니다. 처음에는 철새의 연결 고리의 네트워크를 확인 Wholemount 접근 따라서 체인 마이 그 레이션을 조절 메커니즘에 대한 통찰력을 제공하기 위해 계속됩니다. wholemount 영상으로 VZ과 SVZ 분석 모두 미래 연구와 기존 연구에 대한 우리의 이해를 명확히하는 방법에 대한 새로운 접근법을 추가합니다. 예를 들어, 최근 연구는 성인 SVZ에서 신경 줄기 세포는 뇌실 8 접촉 CD133 + / CD24 - 세포다고 제안했습니다. 섹션에서 immunostaining을 바탕으로,이 작가는 이러한 세포가 multiciliated ependymal 세포의 subpopulation다고 주장했다. 그러나, 전체 ependymal 상피의보다 포괄적인 전망을 제공 wholemount 접근법을 사용하여 우리의 연구에서, 우리는 발견 모든 ependymal 세포 표현 CD24 및 유일한 뇌실 - 접촉 타입 B1의 일부는 + / CD24 -되었습니다 CD133되었습니다 세포를 세포 4. 또한, wholemount 기술은 성인 뇌의 9 신경 줄기 세포의 최근 설명 모자이크 조직 검사 향후 연구에 도움이 될 것을 약속드립니다. 몇몇 연구는 성인 두뇌의 신경 줄기 세포가 균질 인구 아니라는 것을 보여주지만, 지역 지정 및 일반적으로 후각 망울의 interneurons의 특정 subtypes을 생산합니다. 이러한 연구는 신경 줄기 세포의 다른 subpopulations이 특정 전사 요인 10,11,12,13,14 및 / 표현하거나의 지느러미 - 복부와 앞쪽에 - 사후 extents 함께 자신의 지역 로컬 라이제이션을 통해 구분할 수있다 것을 제안 측면 벽 9,15,16. 성인 신경 줄기 세포의 지역 지정 subpopulations 더 많은 분자 마커가 발견됨에 wholemount 영상은 심실 벽을 따라 각 시조 도메인의 parcelation 종합 전망을 제공해야합니다. wholemount 해부 여기에 제시 이미징 기술은 또한 배아에있는 심실 벽을 분석하는 데 사용할 수 있습니다. 배아 측면 벽의 해부는 같은 방식으로, 단계별, 수행됩니다. 난이도에서만 약간의 차이가있다; 배아 심실은 절개가 쉽게 비교적 큰지만, 조직은 조작이 더 어렵게 부드럽고입니다. 특히, 측면 뇌실과 비슷한 노출 엠브리에서 사용할 수OS는 대뇌 피질의 neurogenesis을 공부하는 뇌실의 대뇌 피질의 장벽을 해부하다 수 있습니다. 최근 증거들은 비대칭 centrosome 상속 피질 neurogenesis 17시 심실 표면에서 요골 glia을 유지 제안합니다. 요골 glial 혀끝의 표면 엉 - 얼굴 영상이 나누어 세포 내에서 centrosomes가 불균형 상속하는 방법에 대한 통찰력을 제공할 수 있습니다. 특히 대부분의 기술, 정확한 손재주는 관련된 사람과 마찬가지로 숙달 연습이 필요합니다. 더 나은 결과를 핵심 해부 몇 요소, 그러나,있다 : 1) 조명은 그림자를 만들 수있는 샘플의 조명을 조절하면 같은 포셉를 사용하여) 그렇지 않으면, 2 비교적 균질는 조직의 절개하는 동안 귀중한 콘트라스트를 제공합니다 이 기법에서 두 곤충 핀 포셉 함께 비틀거나 조직을 데리러 사용하지 않습니다하지만), 부드러운 철회의 균형을 3 절단하고 칼을 절단하지 말아야하는 동안 조직을 안정화시키기 위해 지속적으로 재조정 수있는 기동성 핀으로 사용됩니다 단 줄일뿐만 아니라이 절개의 대다수가 실제로에만 간헐적으로 절단과 부드러운 철회를 통해 수행되는 기억, 중간과 측면 벽을 분리하기 위해 부드러운 철회를 제공하는 데 사용됩니다.

the subventricular zone en-face: wholemount staining and ependymal flow

측면 심실의 벽 성인 포유류의 두뇌에서 가장 큰 새싹 지역을 포함하고 있습니다. 이 벽에 subventricular 영역 (SVZ)은 신경 줄기 세포의 행동과 성인 neurogenesis의 규정을 이해하기위한 광범위한 연구의 모델 시스템입니다. 전통적으로,이 연구는 histological 분석을위한 고전 sectioning 기술에 의존합니다. 여기서 우리는 다른 접근이 새싹 지역의 종합 엉 - 얼굴보기를 제공 wholemount 기법을 제시한다. 섹션에 비해 wholemounts는 SVZ 이내에 완료 cytoarchitecture와 세포 관계를 유지하고있다. 이 방법은 최근 성인 신경 줄기 세포 또는 형식 B1 세포는 측면 심실을 안감 차별 ependymal 세포와 혼합 neuroepithelium의 일부입니다 것으로 나타났습니다. 또한이 접근들은 뇌실에서 생성 ependymal 세포와 뇌척수 흐름의 평면 편광을 연구하는 데 사용되었습니다. 성인 신경 줄기 세포가 지역 지정 이기종 인구는 것을 최근에 증거로 wholemount 접근 가능성이 줄기 세포 틈새의 조직과 parcellation을 이해하는 필수적인 도구가 될 것입니다.

Watch this Scientific Journal Video about The Subventricular Zone En-face: Wholemount Staining and Ependymal Flow at JoVE.com

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

측면 뇌실 벽은 성인 포유류의 두뇌에서 가장 큰 새싹 지역을 포함하고 있습니다. 전통적으로이 지역에서 neurogenesis 연구는 histological 분석을위한 고전 sectioning 기술에 의존합니다. 여기서 우리는 다른 접근이 새싹 지역의 종합 엉 - 얼굴보기를 제공 wholemount 기법을 제시한다.

Subventricular 존 엉 - 얼굴 : Wholemount 스테 이닝 및 Ependymal 흐름

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 ...

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Neuroscience

41.6K 조회수.  University of California, San Francisco - UCSF. 측면 뇌실 벽은 성인 포유류의 두뇌에서 가장 큰 새싹 지역을 포함하고 있습니다. 전통적으로이 지역에서 neurogenesis 연구는 histological 분석을위한 고전 sectioning 기술에 의존합니다. 여기서 우리는 다른 접근이 새싹 지역의 종합 엉 - 얼굴보기를 제공 wholemount 기법을 제시한다.

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

Wholemount Immunohistochemistry for Revealing Complex Brain Topography

The repeated and well-understood cellular architecture of the cerebellum make it an ideal model system for exploring brain topography. Underlying its relatively uniform cytoarchitecture is a complex array of parasagittal domains of gene and protein expression. The molecular compartmentalization of the cerebellum is mirrored by the anatomical and functional organization of afferent fibers. To fully appreciate the complexity of cerebellar organization we previously refined a wholemount staining approach for high throughput analysis of patterning defects in the mouse cerebellum. This protocol describes in detail the reagents, tools, and practical steps that are useful to successfully reveal protein expression patterns in the adult mouse cerebellum by using wholemount immunostaining. The steps highlighted here demonstrate the utility of this method using the expression of zebrinII/aldolaseC as an example of how the fine topography of the brain can be revealed in its native three-dimensional conformation. Also described are adaptations to the protocol that allow for the visualization of protein expression in afferent projections and large cerebella for comparative studies of molecular topography. To illustrate these applications, data from afferent staining of the rat cerebellum are included.

Neural circuits are topographically organized into functional compartments with specific molecular profiles. Here, we provide the practical and technical steps for revealing global brain topography using a versatile wholemount immunohistochemical staining approach. We demonstrate the utility of the method using the well-understood cytoarchitecture and circuitry of cerebellum.

의미 심장한 복잡한 두뇌 지형에 대한 Wholemount Immunohistochemistry

The repeated and well-understood cellular architecture of the cerebellum make it an ideal model system for exploring brain topography. Underlying its ...

연구

신경과학

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.

칼슘 이미징에 대한 급성 Subventricular 지대 슬라이스의 작성

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

Neonatal Subventricular Zone Electroporation

Neural stem cells (NSCs) line the postnatal lateral ventricles and give rise to multiple cell types which include neurons, astrocytes, and ependymal cells1. Understanding the molecular pathways responsible for NSC self-renewal, commitment, and differentiation is critical for harnessing their unique potential to repair the brain and better understand central nervous system disorders. Previous methods for the manipulation of mammalian systems required the time consuming and expensive endeavor of genetic engineering at the whole animal level2. Thus, the vast majority of studies have explored the functions of NSC molecules in vitro or in invertebrates.
Here, we demonstrate the simple and rapid technique to manipulate neonatal NPCs that is referred to as neonatal subventricular zone (SVZ) electroporation. Similar techniques were developed a decade ago to study embryonic NSCs and have aided studies on cortical development3,4 . More recently this was applied to study the postnatal rodent forebrain5-7. This technique results in robust labeling of SVZ NSCs and their progeny. Thus, postnatal SVZ electroporation provides a cost and time effective alternative for mammalian NSC genetic engineering.

We demonstrate a minimally invasive technique referred to as neonatal subventricular zone electroporation. The technique consists of injecting plasmid DNA into the lateral ventricles of neonatal pups and applying electrical current to deliver and genetically manipulate neural stem cells

신생아 Subventricular 지대 Electroporation

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

Immunohistochemical and Calcium Imaging Methods in Wholemount Rat Retina

In this paper we describe the tools, reagents, and the practical steps that are needed for: 1) successful preparation of wholemount retinas for immunohistochemistry and, 2) calcium imaging for the study of voltage gated calcium channel (VGCC) mediated calcium signaling in retinal ganglion cells. The calcium imaging method we describe circumvents issues concerning non-specific loading of displaced amacrine cells in the ganglion cell layer.

Immunohistochemistry protocols are used to study the localization of a specific protein in the retina. Calcium imaging techniques are employed to study calcium dynamics in retinal ganglion cells and their axons.

Wholemount 쥐의 망막에서 면역 조직 화학 및 칼슘 이미징 방법

In this paper we describe the tools, reagents, and the practical steps that are needed for: 1) successful preparation of wholemount retinas for ...

Live Imaging of the Ependymal Cilia in the Lateral Ventricles of the Mouse Brain

Multiciliated ependymal cells line the ventricles in the adult brain. Abnormal function or structure of ependymal cilia is associated with various neurological deficits. The current ex vivo live imaging of motile ependymal cilia technique allows for a detailed study of ciliary dynamics following several steps. These steps include: mice euthanasia with carbon dioxide according to protocols of The University of Toledo&#8217;s Institutional Animal Care and Use Committee (IACUC); craniectomy followed by brain removal and sagittal brain dissection with a vibratome or sharp blade to obtain very thin sections through the brain lateral ventricles, where the ependymal cilia can be visualized. Incubation of the brain&#8217;s slices in a customized glass-bottom plate containing Dulbecco&#8217;s Modified Eagle&#8217;s Medium (DMEM)/High-Glucose at 37 &#176;C in the presence of 95%/5% O2/CO2 mixture is essential to keep the tissue alive during the experiment. A video of the cilia beating is then recorded using a high-resolution differential interference contrast microscope. The video is then analyzed frame by frame to calculate the ciliary beating frequency. This allows distinct classification of the ependymal cells into three categories or types based on their ciliary beating frequency and angle. Furthermore, this technique allows the use of high-speed fluorescence imaging analysis to characterize the unique intracellular calcium oscillation properties of ependymal cells as well as the effect of pharmacological agents on the calcium oscillations and the ciliary beating frequency. In addition, this technique is suitable for immunofluorescence imaging for ciliary structure and ciliary protein localization studies. This is particularly important in disease diagnosis and phenotype studies. The main limitation of the technique is attributed to the decrease in live motile cilia movement as the brain tissue starts to die.

Using high-resolution differential interference contrast (DIC) microscopy, an ex vivo observation of the beating of motile ependymal cilia located within the mouse brain ventricles is demonstrated by live-imaging. The technique allows a recording of the unique ciliary beating frequency and beating angle as well as their intracellular calcium oscillation pacing properties.

마우스 뇌의 좌우 심실의 뇌실막 섬모의 라이브 영상

Multiciliated ependymal cells line the ventricles in the adult brain. Abnormal function or structure of ependymal cilia is associated with various ...

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.

셀 성인 마우스 뇌실 영역에서 신경 줄기 및 전구 세포의 정렬과 세포주기 역학의 라이브 이미징

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.

마우스 뇌량 밑 영역에서의 분리 및 성인 신경 줄기 세포의 문화

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

Measuring Post-Stroke Cerebral Edema, Infarct Zone and Blood-Brain Barrier Breakdown in a Single Set of Rodent Brain Samples

One of the most common causes of morbidity and mortality worldwide is ischemic stroke. Historically, an animal model used to stimulate ischemic stroke involves middle cerebral artery occlusion (MCAO). Infarct zone, brain edema and blood-brain barrier (BBB) breakdown are measured as parameters that reflect the extent of brain injury after MCAO. A significant limitation to this method is that these measurements are normally obtained in different rat brain samples, leading to ethical and financial burdens due to the large number of rats that need to be euthanized for an appropriate sample size. Here we present a method to accurately assess brain injury following MCAO by measuring infarct zone, brain edema and BBB permeability in the same set of rat brains. This novel technique provides a more efficient way to evaluate the pathophysiology of stroke.

This protocol describes a novel technique of measuring the three most important parameters of ischemic brain injury on the same set of rodent brain samples. Using only one brain sample is highly advantageous in terms of ethical and economic costs.

설치류 뇌 샘플의 단일 세트에서 뇌졸중 후 뇌 부종, 경색 영역 및 혈액 뇌 장벽 고장 측정

One of the most common causes of morbidity and mortality worldwide is ischemic stroke. Historically, an animal model used to stimulate ischemic stroke ...

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.

정확하고 심도 있는 정량적 프로테아메 분석을 위한 성인 Subependymal 영역의 극저온 단면 해부

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

<meta charset="utf8"></head><body>쥐 뇌실하 영역에서 분리된 NSC의 효율적인 핵 분석

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>저자 스포트라이트: Improved Nucleofection for High-Efficiency Gene Delivery in Murine Subventricular Zone-Derived Neural Stem Cell Cultures

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.

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 ...