وأيد هذا العمل من المنح المقدمة من وزارة الدفاع (فكرة الجائزة التنمية، W81XWH-10-1-0041، AB)، CT الجذعية منحة الخلية (AB)، والمعهد الوطني للصحة NRSA 10668225 (DMF). ويستند المواد موجودة على العمل المدعوم جزئيا من ولاية كونيتيكت تحت الخلايا الجذعية كونيتيكت برنامج المنح البحثية. كان شركة والممولين محتوياته هي الجهة الوحيدة المسؤولة عن أصحابها ولا تعبر بالضرورة عن وجهات النظر الرسمية للدولة من ولاية كونيتيكت، وزارة الصحة العامة في ولاية كونيتيكت أو الابتكارات CT، أي دور في تصميم الدراسة وجمع البيانات والتحليل، وقرار نشر أو إعداد المخطوطة.

هذا الإجراء هو وفقا لمتطلبات IACUC ييل. هل يجوز للعلماء التأكد من أن تتم الموافقة على المبادئ التوجيهية IACUC وتابع وفقا لاحتياجاتها المؤسسية. 1. القسم 1. إعداد الماصات DNA، والحلول، والزجاج <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> توليد عالية النقاء (OD 260/280&gt; 1.80) التركيز العالي (ميكروغرام / ميكرولتر&gt; 2.5) سم داخلي المنشأ خالية من الحمض النووي. </li><li style=";text-align:right;direction:rtl"> إعداد محلول ملحي 0.9٪ وتصفية العقيمة من خلال مرشح ميكرون 22. </li><li style=";text-align:right;direction:rtl"> مكان الحريق 10 سم مصقول أنابيب زجاجية البورسليكات الشعرية (OD: 1.5 مم، ID: 1.10 مم) إلى نموذج PP-830 PC-10 Narishige الزجاج ماصة مجتذب مع سلك kanthal. تعيين مجتذب لسحب خطوة واحدة المرجح في 70،5 ° C. نصائح كسر مع ملقط دائرية وتفتيش تحت المجهر تشريح للحواف خشنة. نصائح شطبة ومكان تحت مصباح الأشعة فوق البنفسجية لمدة 15 دقيقة. يجب وضع علامة ماصات سحبت في 2 مم من حافة الحافة. </li><li style=";text-align:right;direction:rtl"> إعداد 0.1٪ الوزن / الحجم سريع الخضراء solutiعلى طريق خلط محلول معقم المالحة تصفيتها مع الأخضر 0.9٪ سريع الجفاف. </li></ol> 2. القسم 2. إعداد الحيوان، الزجاج ماصة تحميل وحقن البلازميدات <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> إزالة يوم بعد الولادة 0-1 الجراء بشكل فردي من الأقفاص، ومكان على طبق بتري الزجاج قبل المبردة إلى 4 ° C، ثم مكان على الجليد الرطب. </li><li style=";text-align:right;direction:rtl"> بعد حوالي 5 دقائق، تحديد حالة التخدير من خلال الاستفادة من استجابة قرصة القدم. إذا لم يحدث حركة، الجراء خاضعة للحقن داخل البطيني. </li><li style=";text-align:right;direction:rtl"> من المهم أن نلاحظ أن الجمع بين الأخضر بسرعة، DNA، ومحلول ملحي يمكن أن يؤدي إلى التبخر السريع، تبلور، والحصار المفروض على ماصات زجاجية. لذلك، إنشاء محلول المخزون وقسامة 1-2 ميكرولتر على parafilm مباشرة قبل الحقن. </li><li style=";text-align:right;direction:rtl"> نضح وحدة التخزين بالكامل aliquoted مع ماصة الزجاج سحبت. </li><li style=";text-align:right;direction:rtl"> عقد رئيس الجرو بين الإبهام والسبابة من يدك المهيمنة أقل. </li><li style=";text-align:right;direction:rtl"> تشغيل مصباح علىد عقد رئيس الجرو خارج للتو من شعاع ضوء. إذا لزم الأمر، وطرح المالحة على رأسه لتقليل كمية الضوء المنعكس من الجرو. وينبغي الآن أن تكون مضيئة البطينين. </li><li style=";text-align:right;direction:rtl"> مع يدك المهيمنة، أدخل الإبرة في بطين الوحشي الأقرب لك (الشكل 1A و 1B). يجب أن يكون مكان الحقن مسافة واحدة تقريبا من الدرز اللامي والعين و2 مم الوحشي للخياطة السهمي. سحبت إدراج ماصة للعلامة مم 2 لالجرو P0، التي ينبغي ضمان النفاذ إليها من البطين الجانبي. من قبيل الصدفة، قد تشاهد مرة أخرى ملء CSF في ماصة من إطلاق الضغط داخل الجمجمة. لتخفيف الضغط داخل الجمجمة، والذي يساعد في ضخ البلازميد، وتخفيف قبضة الخاص بك على رئيس pup.Step 2.8. حقن حوالي 0.5 ميكرولتر من البلازميد في البطين الجانبي باستخدام محقن الهواء لضغوط (Picospritzer، باركر). </li></ol> 3. الباب 3. الصعق الكهربائي والاسترداد <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> SEر الجهد للمولد الصعق الكهربائي لمدة 5 البقول مربع، و 50 مللي ثانية / 100 فولت في نبض، مع فترات ميللي ثانية 950. وأملت خصوصية المكانية للبلازميد الصعق الكهربائي من قبل اتجاهية نقل المسؤول. وينبغي إيلاء اهتمام وثيق لوضع نظرا لعدم تجانس السكان الخلايا الجذعية على طول SVZ 8. وقد تم تحديد الاختلافات في معدلات الانتشار ومصير الخلية للمواقع بطني-الظهرية والذيلية منقاري، 9،10. </li><li style=";text-align:right;direction:rtl"> مرة واحدة يتم حقن البلازميد، وتراجع أقطاب منتاش في برنامج تلفزيوني. هذه الخطوة هو تعظيم ونقل المسؤول لمنع الحروق الناجمة عن المقاومة. </li><li style=";text-align:right;direction:rtl"> وضع القطب الموجب على جدار الجانبي الظهري للالجرو بالقرب من الأذن المماثل من موقع الحقن بلازميد (الشكل 1C و 1D). وضع قطب كهربائي سلبي على نصف الكرة الجانبية المقابل بطني لخطم الجرو ل. </li><li style=";text-align:right;direction:rtl"> بدء النقل الحالي عن طريق الضغط على يمشي على الأقدام نبضساحرة واكتساح دواسة الأقطاب من الظهرية الجانبية لاستخدام ~ 25 درجة زاوية فترات. </li><li style=";text-align:right;direction:rtl"> electroporated مكان الجراء على وسادة التدفئة لمدة 5 دقائق. تدوير بلطف الجراء كل ثانية 30 مع تحفيز معتدل. </li></ol>

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الكتاب ليس لديهم لجعل الكشف.

نحن هنا التفاصيل أسلوب الصعق الكهربائي SVZ حديثي الولادة، وهي تقنية لبسرعة وبقوة تسمية الخلايا الجذعية والتلاعب SVZ وذريتها. هناك العديد من المزايا التي الصعق الكهربائي وبالمقارنة مع غيرها من التقنيات. أولا، نظرا للوضع العلامات التنسيق من الخلايا، واحد قادر على تمييز...

<table border="1" style=";text-align:right;direction:rtl"><tbody><tr><td> اسم كاشف </td><td> شركة </td><td> كتالوج رقم </td><td> التعليقات (اختياري) </td></tr><tr><td> الأنابيب الثقيلة البورسليكات ملمع </td><td> سوتر الصك </td><td> BF150-110-10 </td><td></td></tr><tr><td> زجاج مزدوج المرحلة Micropipette بولير </td><td> Narishige </td><td> PC-10H </td><td></td></tr><tr><td> سريع الأخضر </td><td> فيشر العلمية </td><td> 0521192205 </td><td></td></tr><tr><td> ECM 830 ساحة مولد موجة النبض </td><td> جهاز هارفارد </td><td> 45-0052 </td><td></td></tr><tr><td> Tweezertrodes </td><td> جهاز هارفارد </td><td> 45-0488 </td><td></td></tr><tr><td> الألياف البصرية ضوء المصدر </td><td> فيشر العلمية </td><td> 12-562-36 </td><td></td></tr><tr><td> التنغستن هالوجينمصباح </td><td> اوشيو أمريكا، وشركة </td><td> 1002247 </td><td></td></tr><tr><td> Picospritzer II </td><td> باركر الصكوك </td><td> 052-0312-900 </td><td></td></tr></tbody></table>

neonatal subventricular zone electroporation

الخلايا الجذعية العصبية (NSCs) خط البطينين الوحشي بعد الولادة وتؤدي إلى أنواع خلايا متعددة والتي تشمل الخلايا العصبية، الخلايا النجمية، وخلايا البطانة العصبية 1. فهم المسارات الجزيئية المسؤولة عن التزام مجلس الأمن القومي، التجديد الذاتي، والتمايز هو أمر حاسم لتسخير إمكاناتها الفريدة لإصلاح الدماغ وفهم أفضل اضطرابات الجهاز العصبي المركزي. مطلوب الأساليب السابقة للتلاعب من نظم الثدييات المسعى تستغرق وقتا طويلا ومكلفة للهندسة الوراثية على مستوى الحيوان كله 2. وهكذا، واستكشفت الغالبية العظمى من الدراسات وظائف الجزيئات NSC في المختبر أو في اللافقاريات. هنا، علينا أن نبرهن على تقنية بسيطة وسريعة لمعالجة المواليد الشخصيات التي يشار اليها على انها حديثي الولادة الصعق الكهربائي subventricular (SVZ) المنطقة. تم تطوير تقنيات مماثلة قبل عقد من الزمن لدراسة NSCs الجنينية ولقد ساعد على دراسات corticaL 3،4 التنمية. وفي الآونة الأخيرة تم تطبيق هذه لدراسة الدماغ الأمامي القوارض بعد الولادة 5-7. هذه التقنية نتائج قوية في وضع العلامات من NSCs SVZ وذريتها. وهكذا، بعد الولادة SVZ الصعق الكهربائي و يوفر التكلفة والوقت البديل الفعال للهندسة الوراثية NSC الثدييات.

الوليد الصعق الكهربائي منطقة subventricular النتائج في وسم الخلايا الدبقية تقريبا كل شعاعي متجاورة مع، الظهرية الظهرية منطقة subventricular الجانبية، والجانبية بعد حركة &quot;الكاسح&quot; من القطب منتاش (الشكل 1E). ومع ذلك، يمكن أن تكون مصممة الصعق الكهربائي لمتطلبات التجربة ...

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

Neonatal Subventricular Zone Electroporation

ونحن يبرهن على وجود تقنية مينيملي ويشار إلى الولدان الصعق الكهربائي منطقة subventricular. تقنية حقن الحمض النووي يتكون من البلازميد إلى البطينين الوحشي للالجراء حديثي الولادة وتطبيق التيار الكهربائي لتقديم والتلاعب وراثيا الخلايا الجذعية العصبية

الوليد الصعق الكهربائي المنطقة Subventricular

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 Views.  Yale University School of Medicine. ونحن يبرهن على وجود تقنية مينيملي ويشار إلى الولدان الصعق الكهربائي منطقة subventricular. تقنية حقن الحمض النووي يتكون من البلازميد إلى البطينين الوحشي للالجراء حديثي الولادة وتطبيق التيار الكهربائي لتقديم والتلاعب وراثيا الخلايا الجذعية العصبية

Video: 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.

المنطقة Subventricular اون الوجه : يلطخ Wholemount والتدفق البطانة العصبية

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 ...

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.

إعداد شرائح الحاد المنطقة Subventricular للتصوير الكالسيوم

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.

حديثي الولادة حنوني التثقيب الكهربائي السطحية

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.

-النوع الفرعي انتقائية Electroporation للمن القشرية Interneurons

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.

فرز خلية العصبية الجذعية وخلايا السلف من الفأر Subventricular المنطقة الكبار وحية على التصوير من حيوية دورة الخلية الخاصة

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.

العزلة والثقافة من الخلايا الجذعية البالغة العصبية من المنطقة تحت الثفني ماوس

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.

تشريح المقطع المبرد للمنطقة تحت البطانية للبالغين من أجل تحليل دقيق وعميق للبروتيوم الكمي

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.

نموذج القوارض الوليدية لحقن الوريد خلف الحجاج

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

Delivering Intrathecal Injections to Neonatal Mice

توصيل الحقن داخل القراب للفئران حديثي الولادة

<meta charset="utf8"></head><body>كفاءة النواة من NSCs المعزولة من منطقة البطين تحت البطين الفئران

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>تسليط الضوء على المؤلف: تحسين النواة لتوصيل الجينات عالية الكفاءة في مزارع الخلايا الجذعية العصبية المشتقة من منطقة البطين تحت البطين

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.

عزل وتوسع ونواة الخلايا الجذعية العصبية من منطقة تحت البطين الفئران البالغة

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 ...