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

<ol>
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<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.22 &#956;m pore-filter bottles (250 mL)</td>
			<td>VWR</td>
			<td>514-0330P</td>
			<td></td>
		</tr>
		<tr>
			<td>0.22 &#956;m pore-filter bottles (500 mL)</td>
			<td>VWR</td>
			<td>514-0332P</td>
			<td></td>
		</tr>
		<tr>
			<td>12-well plate</td>
			<td>LabClinics</td>
			<td>PLC30012</td>
			<td></td>
		</tr>
		<tr>
			<td>15 mL tube</td>
			<td>Fisher</td>
			<td>10738771</td>
			<td></td>
		</tr>
		<tr>
			<td>24-well plate</td>
			<td>LabClinics</td>
			<td>PLC30024</td>
			<td></td>
		</tr>
		<tr>
			<td>4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES)</td>
			<td>BioWest</td>
			<td>L0180-100</td>
			<td></td>
		</tr>
		<tr>
			<td>4&#8242;,6-diamidino-2-phenylindole (DAPI)</td>
			<td>Sigma</td>
			<td>D9542</td>
			<td></td>
		</tr>
		<tr>
			<td>48-well plate</td>
			<td>ThermoFisher</td>
			<td>150687</td>
			<td></td>
		</tr>
		<tr>
			<td>6-well plate</td>
			<td>LabClinics</td>
			<td>PLC30006</td>
			<td></td>
		</tr>
		<tr>
			<td>96-well plate</td>
			<td>Labclinics</td>
			<td>PLC30096</td>
			<td></td>
		</tr>
		<tr>
			<td>Accutase solution</td>
			<td>Sigma</td>
			<td>A6964-100ML</td>
			<td>Referred as enzymatic solution</td>
		</tr>
		<tr>
			<td>Amaxa Mouse Neural Stem Cell Nucleofector Kit</td>
			<td>Lonza</td>
			<td>VPG-1004</td>
			<td></td>
		</tr>
		<tr>
			<td>Amaxa Nucleofector Iib</td>
			<td>Lonza</td>
			<td>10700807</td>
			<td></td>
		</tr>
		<tr>
			<td>Antibiotic/Antimitotic (A/A)</td>
			<td>Sigma</td>
			<td>A5955</td>
			<td></td>
		</tr>
		<tr>
			<td>Apo-t-Transferrine</td>
			<td>Sigma</td>
			<td>T2252-1G</td>
			<td></td>
		</tr>
		<tr>
			<td>Basic Fibroblast Growth Factor (bFGF)</td>
			<td>Sigma</td>
			<td>F0291</td>
			<td></td>
		</tr>
		<tr>
			<td>Blasticidin</td>
			<td>Sigma</td>
			<td>203350</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin (BSA)</td>
			<td>Sigma</td>
			<td>B4287-25MG</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell strainer 40 &#956;m</td>
			<td>LabClinics</td>
			<td>PLC93040</td>
			<td></td>
		</tr>
		<tr>
			<td>Deoxynucleotide triphosphate (dNTPs)</td>
			<td>NZYTech</td>
			<td>MB08701</td>
			<td></td>
		</tr>
		<tr>
			<td>Dimethyl sulfoxide (DMSO)</td>
			<td>Sigma</td>
			<td>D8418</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#8217;s Phosphate Saline Buffer (DPBS)</td>
			<td>Gibco</td>
			<td>14080-055</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Modified Eagle&#39;s Medium (DMEM) F12 1:1</td>
			<td>Gibco</td>
			<td>11320-074</td>
			<td></td>
		</tr>
		<tr>
			<td>E. coli DH5&#945; Competent Cells</td>
			<td>ThermoFisher</td>
			<td>EC0112</td>
			<td></td>
		</tr>
		<tr>
			<td>Earles&#39;s Balanced Salt Solution (EBSS)</td>
			<td>Gibco</td>
			<td>24010-043</td>
			<td></td>
		</tr>
		<tr>
			<td>Epidermal Growth Factor - Human recombinant (EGF)</td>
			<td>Gibco</td>
			<td>53003-018</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethylenediaminetetraacetic acid (EDTA)</td>
			<td>Sigma</td>
			<td>E-6511</td>
			<td></td>
		</tr>
		<tr>
			<td>Fine Forceps</td>
			<td>Fine Science Tools</td>
			<td>11274-20</td>
			<td></td>
		</tr>
		<tr>
			<td>Fine Scissors Sharp</td>
			<td>Fine Science Tools</td>
			<td>14060-09</td>
			<td></td>
		</tr>
		<tr>
			<td>Glucose</td>
			<td>Sigma</td>
			<td>G-7021</td>
			<td></td>
		</tr>
		<tr>
			<td>GoTaq G2 Flexi DNA Polymerase</td>
			<td>Promega</td>
			<td>M7805</td>
			<td>Kit includes reagents for PCR</td>
		</tr>
		<tr>
			<td>Heparin</td>
			<td>Sigma</td>
			<td>H-3149</td>
			<td></td>
		</tr>
		<tr>
			<td>Insuline</td>
			<td>Sigma</td>
			<td>I6634</td>
			<td></td>
		</tr>
		<tr>
			<td>LB agar (Lennox)</td>
			<td>LabKem</td>
			<td>AGLB-00P-500</td>
			<td></td>
		</tr>
		<tr>
			<td>LB broth (Lennox)</td>
			<td>LabKem</td>
			<td>LBBR-00P-500</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Cysteine</td>
			<td>Sigma</td>
			<td>C-8277</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Glutamine</td>
			<td>Gibco</td>
			<td>25030-024</td>
			<td></td>
		</tr>
		<tr>
			<td>Neubauer chamber</td>
			<td>Blaubrand</td>
			<td>BR718620</td>
			<td></td>
		</tr>
		<tr>
			<td>Nuclease free water</td>
			<td>Labbox</td>
			<td>WATR-00A-10K</td>
			<td></td>
		</tr>
		<tr>
			<td>NZYMaxiprep Endotoxin Free Kit</td>
			<td>NZYTech</td>
			<td>MB39901</td>
			<td></td>
		</tr>
		<tr>
			<td>Papain Lyophilized</td>
			<td>Worthington</td>
			<td>LS003119</td>
			<td></td>
		</tr>
		<tr>
			<td>Progesterone</td>
			<td>Sigma</td>
			<td>P-6149</td>
			<td></td>
		</tr>
		<tr>
			<td>Putrescine</td>
			<td>Sigma</td>
			<td>P-7505</td>
			<td></td>
		</tr>
		<tr>
			<td>Scalpel</td>
			<td>Fine Science Tools</td>
			<td>10316-14</td>
			<td></td>
		</tr>
		<tr>
			<td>shSCRAMBLE</td>
			<td>Mission (Sigma)</td>
			<td>SHC003</td>
			<td></td>
		</tr>
		<tr>
			<td>shSNRPN</td>
			<td>Mission (Sigma)</td>
			<td>TRCN0000109285</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Selenite</td>
			<td>Sigma</td>
			<td>S-9133</td>
			<td></td>
		</tr>
		<tr>
			<td>Spatula</td>
			<td>Fine Science Tools</td>
			<td>10090-17</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile PES Syringe Filters (0.22 &#956;m pore-filter)</td>
			<td>Epica</td>
			<td>SFPE-22E-050</td>
			<td></td>
		</tr>
		<tr>
			<td>T12.5 cm2 Flask</td>
			<td>Biofil</td>
			<td>TCF012025</td>
			<td></td>
		</tr>
		<tr>
			<td>T25 cm2 Flask</td>
			<td>LabClinics</td>
			<td>PLC70025</td>
			<td></td>
		</tr>
		<tr>
			<td>T75 cm2 Flask</td>
			<td>LabClinics</td>
			<td>PLC70075</td>
			<td></td>
		</tr>
		<tr>
			<td>Tweezers</td>
			<td>Fine Science Tools</td>
			<td>91150-20</td>
			<td></td>
		</tr>
	</tbody>
</table>

isolation, expansion, and nucleofection of neural stem cells from adult murine subventricular zone

Neural stem cells (NSCs) are multipotent stem cells resident in the brain. These cells possess the ability to self-renew and differentiate into the three neural lineages: astrocytes, oligodendrocytes, and neurons1. Consequently, NSCs play a crucial role in adult neurogenesis in mammals, a process where new neurons are generated in the brain2. NSCs predominantly reside in two regions within the adult brain termed neurogenic niches: the subventricular zone (SVZ) along the walls of the lateral ventricles and the subgranular zone (SGZ) within the dentate gyrus of the hippocampus3,4. NSCs (also known as B cells) in the SVZ, the most active neurogenic niche in the adult mouse, self-renew and produce transit-amplifying progenitors (TAPs or C cells) that subsequently differentiate into neuroblasts (A cells). These neuroblasts migrate through the rostral migratory stream (RMS) to the olfactory bulbs (OB), where they undergo full differentiation into interneurons, integrating into the pre-existing circuitry3,4,5,6.
Understanding the intricate interplay of molecular cues and signals that regulate NSCs within these niches is important to harness their potential for therapeutic applications. For that purpose, various methods have been developed to study this cell population, ranging from selective primary culture of NSCs to cell selection using surface markers7,8,9,10. The present manuscript details the isolation and culturing of SVZ NSCs in vitro using a serum-free selective medium containing both mitogens: basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF). This medium facilitates cell proliferation and maintains the stemness of NSCs obtained from the SVZ of adult mouse&#160;brains forming in vitro clonal, three-dimensional, non-adherent aggregates known as neurospheres9. Neurosphere cultures serve as a controlled platform for manipulating and studying the molecular mechanisms and factors affecting NSC proliferation, self-renewal, differentiation, and survival11. Notably, the number of primary neurospheres formed in the culture allows an estimation of the number of NSCs present in the SVZ in vivo, making it a powerful tool for studying the effects of different conditions on the adult NSC pool12,13. Furthermore, once the primary culture is established, NSCs can generate new aggregates (secondary neurospheres) upon passaging through symmetric divisions under proliferation conditions14. Thus, low-density seeding of secondary neurosphere cells (clonal assay) can be utilized to assess the self-renewal rate of these cultures4,15,16,17.
Despite the potential of neurospheres in uncovering the mechanisms governing NSC regulation, some researchers question the validity of in vitro findings, arguing that the artificial conditions in which cells grow might not faithfully replicate the intricate in vivo microenvironment of neurogenic niches18,19,20,21,22. Another controversial point revolves around the observed heterogeneity in neurospheres. Nonetheless, this variability is believed to mirror the symmetric and asymmetric divisions of the NSCs that naturally occur in vivo23,24. Furthermore, recent validation has supported the utilization of NSC cultures to predict mechanisms operating within the SVZ neurogenic niche in vivo, and several studies have demonstrated that NSCs cultured in vitro accurately maintain the transcriptomic profile observed in vivo11,25.
Therefore, neurosphere cultures not only serve as a method to explore NSC proliferation and differentiation abilities, but also offer a system to study the influence of genes governing NSC biology. A pivotal technique for investigating gene function in NSCs is gene expression perturbation. siRNAs or genes delivered through plasmids can be transfected into cell cultures, resulting in knockdown or upregulation of the target gene. This versatile approach significantly reduces the time and cost compared to establishing cultures using conditional knockout mice, presenting a promising avenue for unraveling the genetic bases of neurogenesis and exploring therapeutic prospects. Altering the expression of specific genes in NSCs enables the modulation of their behavior, influencing crucial biological processes such as proliferation, differentiation, and migration. However, the prospect of transfecting NSCs, particularly within mouse neurospheres, presents notable challenges. The three-dimensional structure of neurospheres compromises the efficiency of transfection, often resulting in low rates of successful exogenous nucleic acid delivery, which limits the extent of genetic manipulation26,27. Additionally, transfection procedures can detrimentally affect cell viability and functionality27. In this context, we present the nucleofection system as a method to mitigate cell damage, achieving a high survival rate and ensuring higher efficacy in gene delivery assays used to perturb NSC cultures.
This manuscript aims to illustrate the procedure for isolating, expanding, and nucleofecting NSCs from the adult SVZ neurogenic niche to perturb genes using the neurosphere culture system. This method surpasses the effectiveness of traditional transfection protocols, presenting significantly higher survival rates and improved gene delivery efficiency among the targeted cells.

<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

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

Our research is focused on studying how the neural stem cell population is regulated in the adult mammalian brain. Understanding the intrinsic and extrinsic molecular mechanisms that regulate neural stem cells within the neurogenic niches is important to comprehend the biology and to develop future potential therapeutic applications. To study neural stem cell behavior, both in vivo and in vitro approaches are widely used.In vitro neural stem cell cultures offer controlled environments and the possibility to easily manipulate and monitor this population of cells. Techniques for gene expression preservation in vitro are a versatile approach to study molecular mechanisms governing cell biology. However, an efficient and reproducible method for overexpressing and knocking down candidate genes in neural stem cells is still challenging in the field.Traditional transfection methods for gene delivery have proven effective in central nervous system cells. Moreover, these methods affect cell viability and functionality. Thus, refinement of alternative approaches is crucial to manipulate gene expression in neural stem cells.With this protocol, we present an improved nucleofection system that achieves high efficiency of gene delivery in neural stem cell cultures from the adult murine subventricular zone, along with survival rates higher than 80%

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 ...

isolation-expansion-nucleofection-neural-stem-cells-from-adult-murine

Research

JoVE Journal

Neuroscience

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

778 次观看.  Universitat de València. 我们的研究重点是研究神经干细胞群在成年哺乳动物大脑中是如何被调节的。了解在神经源性生态位内调节神经干细胞的内在和外在分子机制对于理解生物学和开发未来潜在的治疗应用非常重要。为了研究神经干细胞的行为，体内和体外方法都被广泛使用。体外神经干细胞培养提供了受控环境，并且可以轻松操作和监测该细胞群。体外基因表达...