Name: in vivo direct reprogramming of resident glial cells into interneurons by intracerebral injection of viral vectors
Uploaded: 2019-06-17T15:00:00
Duration: 12 min 26 s
Description: Watch this Scientific Journal Video about In Vivo Direct Reprogramming of Resident Glial Cells into Interneurons by Intracerebral Injection of Viral Vectors at JoVE.com

<p class="jove_content">Marcella Birtele 由欧盟 Horizon 2020 计划 (H2020-MSCA-ITN-2015) 资助,该计划由玛丽·斯科多夫斯卡-库里埃创新培训网络和第 676408 号赠款协议提供。丹尼尔拉·赖兰德·奥托松由瑞典研究理事会资助(2017-01234)。</p>

<ol>
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K., Zolotukhin, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Helper-free+Production+of+Laboratory+Grade+AAV+and+Purification+by+Iodixanol+Density+Gradient+Centrifugation.">Helper-free Production of Laboratory Grade AAV and Purification by Iodixanol Density Gradient Centrifugation.</a> <em style='font-style: italic'>Molecular Therapy Methods &#38; Clinial Development</em>. <b>10</b>, 1-7 (2018).</li><li>Torper, O., 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=Generation+of+induced+neurons+via+direct+conversion+in+vivo.">Generation of induced neurons via direct conversion in vivo.</a> <em style='font-style: italic'>Proceedings of the National Academy of Sciences of the United States of America</em>. <b>110</b> (17), 7038-7043 (2013).</li><li>Kawaguchi, Y., Kubota, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Correlation+of+physiological+subgroupings+of+nonpyramidal+cells+with+parvalbumin-+and+calbindinD28k-immunoreactive+neurons+in+layer+V+of+rat+frontal+cortex.">Correlation of physiological subgroupings of nonpyramidal cells with parvalbumin- and calbindinD28k-immunoreactive neurons in layer V of rat frontal cortex.</a> <em style='font-style: italic'>Journal of Neurophysiology</em>. <b>70</b> (1), 387-396 (1993).</li><li>Grealish, S., 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=The+A9+dopamine+neuron+component+in+grafts+of+ventral+mesencephalon+is+an+important+determinant+for+recovery+of+motor+function+in+a+rat+model+of+Parkinson&#39;s+disease.">The A9 dopamine neuron component in grafts of ventral mesencephalon is an important determinant for recovery of motor function in a rat model of Parkinson&#39;s disease.</a> <em style='font-style: italic'>Brain</em>. <b>133</b> (Pt 2), 482-495 (2010).</li><li>Thompson, L., Barraud, P., Andersson, E., Kirik, D., Bjorklund, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+dopaminergic+neurons+of+nigral+and+ventral+tegmental+area+subtypes+in+grafts+of+fetal+ventral+mesencephalon+based+on+cell+morphology,+protein+expression,+and+efferent+projections.">Identification of dopaminergic neurons of nigral and ventral tegmental area subtypes in grafts of fetal ventral mesencephalon based on cell morphology, protein expression, and efferent projections.</a> <em style='font-style: italic'>Journal of Neuroscience</em>. <b>25</b> (27), 6467-6477 (2005).</li></ol>

<p class="jove_content">作者没有什么可透露的。</p>

<p class="jove_content">在活体内,可以直接重新编程,可以使用AAV FLEX矢量在Cre-表达小鼠菌株。需要注意的是,小鼠菌株在重新编程功效方面存在差异。对于在纹状体中重新编程,NG2-Cre 小鼠线已证明比其他菌株更有效。在开始使用新的动物菌株之前,请务必检查小鼠提供者指南,随着时间的推移,Cre表达,因为动物的年龄经常影响这种蛋白质表达的特异性。在我们的研究中,超过12周的动物没有用于体内重新编程,因为NG2胶质以外的细胞存在Cre表达的风险。建议对只注射Synapsin-FLEX-GFP结构的控制动物进行持续存在和监测。这允许监测动物的GFP阳性细胞,如果没有重新编程基因(ALN),不应该存在。</p>
<p class="jove_content">为了识别新重新编程的神经元,一种神经元特定的识别方法,如该协议中描述的方法至关重要。这允许正确识别和区分重新编程的神经元从内源性周围细胞,这是特别相关的,当重编程在同源区域。</p>
<p class="jove_content">目标为病毒注射的正确结构也很重要。因此,病毒注射的立体手术很重要,需要准确处理,特别是在针对大脑较小结构时。</p>
<p class="jove_content">我们之前已经表明,<sup class="xref"></sup>基于使用相同重新编程因子的体外重新编程实验预测体内重新编程的结果是不可靠的。因此,所有感兴趣的因素都需要在体内进行测试。在我们手中,许多不同的因子组合给予相同的神经元亚型(即体内的内神经元11),尽管这些基因与其他神经元<sup class="xref"></sup>的发展有关。</p>
<p class="jove_content">重新编程神经元的全细胞贴片夹是一种精细的技术,组织处理对于良好的结果非常重要。用冰冷的克雷布斯溶液灌注可改善组织质量。此外,修补的神经元需要仔细处理。即使重新编程的神经元的成熟和型型特征可以通过全细胞贴片夹进行一些评估,这些细胞也不能完全与内源性神经元进行比较。应使用其他类型的分析,如基因组测序(例如RNA测序)来进一步确认重新编程的细胞身份。</p>
<p class="jove_content">本文描述的技术可以考虑发展未来治疗,其中神经元替代需要在大脑中。虽然在体内重新编程还处于早期阶段,而且人类的翻译尚未预见到,但这项技术可以提供一种评估大脑外源基因功能和研究体内细胞成熟的方法。</p>

<p class="jove_content">该方法的总体目标是有效地将体内的大脑驻留胶质转化为功能性和亚型特异性神经元,如帕夫巴平表达内神经元。这为开发脑病替代疗法提供了一步,无需外源细胞源。它还创造了一个工具,研究细胞命运开关就地。</p>
<p class="jove_content">大脑产生新神经元的能力有限。因此,在神经系统疾病中,需要外源细胞源来修复大脑。为此,多年来,不同的细胞来源都进行了紧张的研究,包括来自原发组织的细胞、干细胞衍生细胞和重新编程的细胞1、2、3。<sup class="xref"></sup><sup></sup><sup class="xref"></sup><sup></sup><sup class="xref"></sup>将常驻脑细胞直接重新编程到神经元是最近的一种方法,它可以提供一个有吸引力的方法来修复大脑,因为它使用病人自己的细胞在大脑内产生新的神经元。迄今为止,已有几份报告显示,在体内通过病毒载体在大脑4、5、6、7<sup class="xref"></sup><sup></sup><sup class="xref"></sup><sup></sup><sup class="xref"></sup><sup></sup><sup class="xref"></sup><sup class="xref"></sup><sup></sup><sup class="xref"></sup> 8、9的不同大脑区域进行病毒载体的重新编程,如皮层、脊髓、纹状体和中脑5、10、11,<sup class="xref"></sup><sup></sup><sup class="xref"></sup>以及<sup class="xref"></sup><sup></sup>完整和病变<sup class="xref"></sup><sup></sup><sup class="xref"></sup><sup></sup>的大脑5、8、11、12。<sup class="xref"></sup><sup></sup><sup class="xref"></sup>抑制神经元和兴奋神经元都获得了4,8,但这些细胞的精确<sup class="xref"></sup><sup></sup><sup class="xref"></sup>表型或功能尚未得到详细分析。</p>
<p class="jove_content">在此协议中,我们描述了对体内重新编程神经元的更有效的重新编程和细胞特异性识别。我们提供基于功能和免疫组织化学特征的神经元成熟和表型表型表征功能评估方案。</p>
<p class="jove_content">我们使用可诱导的AAV矢量和GFP报告器来识别体内重新编程的神经元。这种病毒载体的选择具有感染大脑分裂细胞和非分裂细胞的优点,增加靶向细胞的数量,作为使用逆转录病毒7,8的替代。<sup class="xref"></sup><sup></sup><sup class="xref"></sup>神经元特异性突触蛋白驱动的FLEX报告器(GFP),使我们能够专门检测新生成的神经元。以前的研究已经使用亚型特异性启动子进行体内重新编程7,9,也允许在特定的细胞类型中表达重新编程的基因和报告器。<sup class="xref"></sup><sup></sup><sup class="xref"></sup>然而,该方法需要进一步识别重新编程的神经元,通过死后分析报告和神经元标记的共同表达。使用神经元特定的报告器(如本文所述)允许直接识别。这提供了成功转换的直接证明,并允许修补夹电生理学所需的活细胞识别。</p>

<table>
	<tbody>
		<tr>
			<td><strong>REAGENTS FOR AAV5 CLONING AND VIRAL VECTOR PREPARATION</strong></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>pAAV-CA-FLEX</td>
			<td>AddGene</td>
			<td>38042</td>
			<td></td>
		</tr>
		<tr>
			<td>Ascl1</td>
			<td>AddGene</td>
			<td>67291</td>
			<td>NM_008553.4</td>
		</tr>
		<tr>
			<td>Lmx1a</td>
			<td>AddGene</td>
			<td>33013</td>
			<td>NM_0033652.5</td>
		</tr>
		<tr>
			<td>Nurr1</td>
			<td>AddGene</td>
			<td>35000</td>
			<td>NM_013613.2</td>
		</tr>
		<tr>
			<td>GFP-syn</td>
			<td>AddGene</td>
			<td>30456</td>
			<td></td>
		</tr>
		<tr>
			<td>LoxP (FLEX) sequence 1</td>
			<td></td>
			<td></td>
			<td>GATCTccataacttcgtataaagtatcctatac<br/>gaagttatatcaaaataggaagaccaatgcttc<br/>accatcgacccgaattgccaagcatcaccatcg<br/>acccataacttcgtataatgtatgctatacgaa<br/>gttatactagtcccgggaaggcgaagacgcgga<br/>agaggctctaga</td>
		</tr>
		<tr>
			<td>LoxP (FLEX) sequences 2</td>
			<td></td>
			<td></td>
			<td>tactagtataacttcgtataggatactttatac<br/>gaagttatcattgggattcttcctattttgatc<br/>caagcatcaccatcgaccctctagtccacatct<br/>caccatcgacccataacttcgtatagcatacat<br/>tatacgaagttatgtccctcgaagaggttcgaa<br/>ttcgtttaaacGGTACCCTCGAC</td>
		</tr>
		<tr>
			<td>pDP5</td>
			<td>Plasmid Factory</td>
			<td>PF435</td>
			<td></td>
		</tr>
		<tr>
			<td>pDP6</td>
			<td>Plasmid Factory</td>
			<td>PF436</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate-Buffered Saline (PBS)</td>
			<td>Thermo Fisher Scientific</td>
			<td>10010023</td>
			<td></td>
		</tr>
		<tr>
			<td>FBS (Fetal bovine serum)</td>
			<td>Thermo Fisher Scientific</td>
			<td>10500064</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin streptomycin</td>
			<td>Thermo Fisher Scientific</td>
			<td>15140122</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM (Dulbecco&#39;s Modified Eagle Medium)+ Glutamax</td>
			<td>Thermo Fisher Scientific</td>
			<td>61965026</td>
			<td></td>
		</tr>
		<tr>
			<td>DPBS (Dulbecco&#39;s Phosphate Buffer Saline)</td>
			<td>Thermo Fisher Scientific</td>
			<td>14190094</td>
			<td></td>
		</tr>
		<tr>
			<td>HEK293 cells</td>
			<td>Thermo Fisher Scientific</td>
			<td>85120602-1VL</td>
			<td></td>
		</tr>
		<tr>
			<td>Flasks</td>
			<td>BD Falcon</td>
			<td>10078780</td>
			<td>175 cm2</td>
		</tr>
		<tr>
			<td>Tris H-CL</td>
			<td>Sigma Aldrich</td>
			<td>10812846001</td>
			<td>
			<div>
			<div>For TE buffer use 10 mM, pH 8.0; for lysis buffer use 50mM, pH 8.5; for IE buffer use 20 mM, pH 8.0; for elution buffer use 20 mM, pH 8.0</div>
			</div>
			</td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>EDTA: Invitrogen</td>
			<td>EDTA: AM9260G</td>
			<td>
			<div>
			<div>For TE buffer use 1 mM EDTA</div>
			</div>
			</td>
		</tr>
		<tr>
			<td>Ultrapure water</td>
			<td></td>
			<td></td>
			<td>
			<div>
			<div>see Ultrapure water system</div>
			</div>
			</td>
		</tr>
		<tr>
			<td>CaCl2</td>
			<td>SigmaAldrich</td>
			<td>C5080</td>
			<td>2.5 M</td>
		</tr>
		<tr>
			<td>Dulbecco&#180;s phosphate-buffered saline (DPBS)</td>
			<td>Thermo Fisher Scientific</td>
			<td>14190136</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Sigma-Aldrich</td>
			<td>S3014</td>
			<td>
			<div>
			<div>FOR HBS use 140 mM; for Lysis Buffer use 150 mM; for IE buffer use 15 mM; for elution buffer use 250 mM</div>
			</div>
			</td>
		</tr>
		<tr>
			<td>MgCl2</td>
			<td>Sigma-Aldrich</td>
			<td>M8266-100G</td>
			<td>
			<div>
			<div>For lysis Buffer: 1 mM</div>
			</div>
			</td>
		</tr>
		<tr>
			<td>Ultracentrifuge sealing tubes</td>
			<td>Beckman Coulter</td>
			<td>Quick-Seal&#174;&#160;Polypropylene Tube</td>
			<td></td>
		</tr>
		<tr>
			<td>OptiPrep&#8482;&#160;Density Gradient Medium</td>
			<td>Sigma Aldrich</td>
			<td>D1556-250ML</td>
			<td></td>
		</tr>
		<tr>
			<td>10-mL syringe-18G needle</td>
			<td>BD</td>
			<td>305064</td>
			<td></td>
		</tr>
		<tr>
			<td>Laboratory glass bottles</td>
			<td>VWR</td>
			<td>?</td>
			<td></td>
		</tr>
		<tr>
			<td>Anion exchange filter</td>
			<td>PALL laboratory</td>
			<td>MSTG25Q6</td>
			<td>
			<div>
			<div>Acrodisc unit with Mustang Q membrane</div>
			</div>
			</td>
		</tr>
		<tr>
			<td>Centrifugal filter unit</td>
			<td>Merck</td>
			<td>Z740210-24EA</td>
			<td>
			<div>
			<div>Amicon Ultra-4 device</div>
			</div>
			</td>
		</tr>
		<tr>
			<td>Endotoxin-Free Plasmid DNA Isolation Kits</td>
			<td>Thermo Fisher Scientific</td>
			<td>A33073</td>
			<td></td>
		</tr>
		<tr>
			<td>Na2PO4</td>
			<td>Sigma-Aldrich</td>
			<td>S7907</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Sigma-Aldrich</td>
			<td>H7523</td>
			<td>15 mL</td>
		</tr>
		<tr>
			<td>Falcon tube</td>
			<td>Thermo Fisher Scientific</td>
			<td>Corning 352196</td>
			<td></td>
		</tr>
		<tr>
			<td>Falcon tube</td>
			<td>Thermo Fisher Scientific</td>
			<td>Corning 352070</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass Vials</td>
			<td>Novatech</td>
			<td>30209-1232</td>
			<td>
			<div>
			<div>CGGCCTCAGFGAGCGA</div>
			</div>
			</td>
		</tr>
		<tr>
			<td>Forward Primer for Inverted Terminal Repeat (ITR) sequence</td>
			<td></td>
			<td></td>
			<td>
			<div>
			<div>GGAACCCCTAGTGATGGAGTT</div>
			</div>
			</td>
		</tr>
		<tr>
			<td>Reverse Primer for Inverted Terminal Repeat (ITR) sequence</td>
			<td></td>
			<td></td>
			<td>
			<div>
			<div>CACTCCCTCTCTGCGCGCTCG</div>
			</div>
			</td>
		</tr>
		<tr>
			<td>5&#180;FAM / 3&#180;BHQ1 probe</td>
			<td>Jena Bioscience</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>0.22 mm filter</td>
			<td>Merck</td>
			<td>SLGV004SL</td>
			<td>Millex-GV filter</td>
		</tr>
		<tr>
			<td><strong>EQUIPMENT AAV5 VIRAL VECTOR PREPARATION</strong></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Freezer -20 &#176;C</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Freezer -80 &#176;C</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Fridge +4 &#176;C</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>AAV viral room</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Ultrapure Water system</td>
			<td>Merck</td>
			<td>Milli-Q&#174; IQ 7000</td>
			<td></td>
		</tr>
		<tr>
			<td>Vortex mixer</td>
			<td>VWR</td>
			<td>444-0004</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultracentrifuge</td>
			<td>Beckman Coulter</td>
			<td>Beckman Optima LE-80K Ultracentrifuge</td>
			<td></td>
		</tr>
		<tr>
			<td>Autoclave</td>
			<td>Tuttnauer</td>
			<td>2540 EL</td>
			<td></td>
		</tr>
		<tr>
			<td>Polymerase Chain Reaction (PCR)</td>
			<td>BioRad</td>
			<td>C1000 Touch Thermal Cycler</td>
			<td></td>
		</tr>
		<tr>
			<td>Quantitative PCR (qPCR)</td>
			<td>Roche</td>
			<td>LightCycler&#174; 480 System</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>Thermo Fisher Scientific</td>
			<td>Sorvall ST16</td>
			<td></td>
		</tr>
		<tr>
			<td>Water Bath</td>
			<td>Thermo Fisher Scientific</td>
			<td>TSGP02</td>
			<td></td>
		</tr>
		<tr>
			<td><strong>ANIMAL MODEL</strong></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>NG2-Cre mice</td>
			<td>Jackson</td>
			<td>NG2-CrexB6129, Stock #008533</td>
			<td></td>
		</tr>
		<tr>
			<td><strong>REAGENTS FOR INJECTION OF REPROGRAMMING FACTORS INTO THE BRAIN</strong></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Water</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Saline</td>
			<td>Apoteket AB</td>
			<td></td>
			<td>70%</td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>Solveco</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Isolfurane</td>
			<td>Apoteket AB</td>
			<td></td>
			<td>
			<div>
			<div>Dilute to 1% solution with warm water.</div>
			</div>
			</td>
		</tr>
		<tr>
			<td>Virkon</td>
			<td>Viroderm</td>
			<td>7511&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Pentobarbital</td>
			<td>Apoteket AB</td>
			<td>P0500000</td>
			<td>
			<div>
			<div>i.p. for terminal procedure at the dose of 60 mg/ml.</div>
			</div>
			</td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>Merck</td>
			<td>S0389-500G</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypan Blu</td>
			<td>Thermo Fisher Scientific</td>
			<td>15250061</td>
			<td></td>
		</tr>
		<tr>
			<td>Retrobeads</td>
			<td>Lumafluor</td>
			<td>R170</td>
			<td></td>
		</tr>
		<tr>
			<td>Buprenorphine</td>
			<td>Apoteket AB</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td><strong>EQUIPMENT FOR INJECTION OF REPROGRAMMING FACTORS INTO THE BRAIN</strong></td>
			<td></td>
			<td></td>
			<td>
			<div>
			<div>From RSG Solingen.</div>
			</div>
			</td>
		</tr>
		<tr>
			<td>Scissors</td>
			<td>VWR</td>
			<td>233-1552</td>
			<td>From Biochem.</td>
		</tr>
		<tr>
			<td>Tweezers</td>
			<td>VWR</td>
			<td>232-0007</td>
			<td></td>
		</tr>
		<tr>
			<td>Forcep</td>
			<td>VWR</td>
			<td>232-0120</td>
			<td></td>
		</tr>
		<tr>
			<td>Scalpel holder</td>
			<td>VWR</td>
			<td>RSGA106.621</td>
			<td>Number 20.</td>
		</tr>
		<tr>
			<td>Scalpel</td>
			<td>VWR</td>
			<td>RSGA106.200</td>
			<td></td>
		</tr>
		<tr>
			<td>Stereotaxic frame</td>
			<td>Stoelting Europe</td>
			<td>51500D</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse ear bars</td>
			<td>Stoelting Europe</td>
			<td>51648</td>
			<td>5 ul</td>
		</tr>
		<tr>
			<td>Syringe with Removable needle</td>
			<td>Hamilton Company</td>
			<td>65</td>
			<td>
			<div>
			<div>0,75 inner diameter and 1.5 outer diameter.</div>
			</div>
			</td>
		</tr>
		<tr>
			<td>Glass capilaries</td>
			<td>Stoelting</td>
			<td>50811</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass capillary puller</td>
			<td>Sutter company</td>
			<td>P-1000</td>
			<td></td>
		</tr>
		<tr>
			<td>Dental drill</td>
			<td>Agnthos AB</td>
			<td>1464</td>
			<td></td>
		</tr>
		<tr>
			<td>Shaver</td>
			<td>Agnthos AB</td>
			<td>GT420</td>
			<td></td>
		</tr>
		<tr>
			<td>Isoflurane Chamber and pump</td>
			<td>Agnthos AB</td>
			<td>8323101</td>
			<td>2 mL</td>
		</tr>
		<tr>
			<td>Syringes</td>
			<td>Merck</td>
			<td>Z118400-30EA</td>
			<td>25G</td>
		</tr>
		<tr>
			<td>Needles</td>
			<td>Merck</td>
			<td>Z192414&#160;Aldrich</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse and neonatal rat adaptor for stereotaxic fram</td>
			<td>Stoelting</td>
			<td>51625</td>
			<td></td>
		</tr>
		<tr>
			<td>Heating pad</td>
			<td>Braintree scientific, inc</td>
			<td>53800M</td>
			<td>
			<div>
			<div>From Covidien 2187.</div>
			</div>
			</td>
		</tr>
		<tr>
			<td>Cotton gauze</td>
			<td>Fisher Scientific</td>
			<td>22-037-902</td>
			<td></td>
		</tr>
		<tr>
			<td>Rubber tube</td>
			<td>Elfa Distrelec</td>
			<td>HFT-A-9.5/4.8 PO-X BK 150</td>
			<td></td>
		</tr>
		<tr>
			<td>Cotton swabs</td>
			<td>Fisher Scientific</td>
			<td>18-366-473</td>
			<td></td>
		</tr>
		<tr>
			<td><strong>REAGENTS FOR WHOLE-CELL PATCH CLAMP RECORDINGS</strong></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Sigma-Aldrich</td>
			<td>S3014</td>
			<td></td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>Sigma-Aldrich</td>
			<td>P9333</td>
			<td></td>
		</tr>
		<tr>
			<td>NaH<sub>2</sub>PO<sub>4</sub>-H<sub>2</sub>O</td>
			<td>Sigma-Aldrich</td>
			<td>S9638</td>
			<td></td>
		</tr>
		<tr>
			<td>MgCl<sub>2</sub>-6 H<sub>2</sub>O</td>
			<td>Sigma-Aldrich</td>
			<td>M2670</td>
			<td></td>
		</tr>
		<tr>
			<td>CaCl<sub>2</sub>-6 H<sub>2</sub>O</td>
			<td>Sigma-Aldrich</td>
			<td>C8106</td>
			<td></td>
		</tr>
		<tr>
			<td>MgSO4-7 H<sub>2</sub>O</td>
			<td>Sigma-Aldrich</td>
			<td>230391</td>
			<td></td>
		</tr>
		<tr>
			<td>NaHCO<sub>3</sub></td>
			<td>Sigma-Aldrich</td>
			<td>S5761</td>
			<td></td>
		</tr>
		<tr>
			<td>Glucose</td>
			<td>Sigma-Aldrich</td>
			<td>G7021</td>
			<td>
			<div>
			<div>see Ultrapure Water system</div>
			</div>
			</td>
		</tr>
		<tr>
			<td>Ultrapure Water</td>
			<td></td>
			<td></td>
			<td>Prepare 1 or 2M.</td>
		</tr>
		<tr>
			<td>KOH</td>
			<td>Sigma-Aldrich</td>
			<td>P5958-500G</td>
			<td></td>
		</tr>
		<tr>
			<td>K-D-gluconate</td>
			<td>Sigma-Aldrich</td>
			<td>G4500&#160;Sigma</td>
			<td></td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>Sigma-Aldrich</td>
			<td>P9541</td>
			<td></td>
		</tr>
		<tr>
			<td>KOH-EGTA (Etilene glycol-bis-N-tetracetic acid)</td>
			<td>Sigma-Aldrich</td>
			<td>E3889&#160;Sigma</td>
			<td></td>
		</tr>
		<tr>
			<td>KOH- Hepes acid (N-2-hydroxyethylpiperazine-N&#8217;-2-ethanesulfonic acid)</td>
			<td>Sigma-Aldrich</td>
			<td>H7523</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Sigma-Aldrich</td>
			<td>S3014</td>
			<td></td>
		</tr>
		<tr>
			<td>Mg2ATP</td>
			<td>Sigma-Aldrich</td>
			<td>A9187</td>
			<td></td>
		</tr>
		<tr>
			<td>Na3GTP</td>
			<td>Sigma-Aldrich</td>
			<td>G8877</td>
			<td></td>
		</tr>
		<tr>
			<td>Biocytin</td>
			<td>Sigma-Aldrich</td>
			<td>B4261</td>
			<td></td>
		</tr>
		<tr>
			<td>Picrotoxin</td>
			<td>Merck</td>
			<td>P1675&#160;Sigma</td>
			<td></td>
		</tr>
		<tr>
			<td>CNQX</td>
			<td>Merck</td>
			<td>C239&#160;Sigma</td>
			<td></td>
		</tr>
		<tr>
			<td>Ice</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td><strong>EQUIPMENT FOR WHOLE-CELL PATCH CLAMP RECORDINGS</strong></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Borosilicate glass pipette</td>
			<td>Sutter Company</td>
			<td>B150-86-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass capillary puller</td>
			<td>Sutter company</td>
			<td>P-1000</td>
			<td></td>
		</tr>
		<tr>
			<td>Vibratome</td>
			<td>Leica</td>
			<td>Leica VT1000 S</td>
			<td></td>
		</tr>
		<tr>
			<td>WaterBath</td>
			<td>Thermo Fisher Scientific</td>
			<td>TSGP02</td>
			<td></td>
		</tr>
		<tr>
			<td>Clampfit software</td>
			<td>Molecular Devices</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Multiclamp software</td>
			<td>Molecular Devices</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td><strong>REAGENTS FOR IMMUNOHISTOCHEMISTRY</strong></td>
			<td></td>
			<td></td>
			<td>
			<div>
			<div>Use at a concentration of 4%.&#160;CAUTION:&#160;PFA is a potent fixative. Avoid ingestion and contact with skin.</div>
			</div>
			</td>
		</tr>
		<tr>
			<td>Paraformaldehyde (PFA)</td>
			<td>Merck Millipore</td>
			<td>1040051000</td>
			<td>
			<div>
			<div>Use at a concentration of 0.1%.</div>
			</div>
			</td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Fisher Scientific</td>
			<td>10254640</td>
			<td>
			<div>
			<div>Donkey.Use at a concentration of 1 : 400.</div>
			</div>
			</td>
		</tr>
		<tr>
			<td>Serum</td>
			<td>Merck Millipore</td>
			<td>S30-100ML</td>
			<td>
			<div>
			<div>Reconstitute the powder in Milli-Q water to 1 mg/mL. Aliquot and store at -20&#176;C,&#160;light sensitive. Use at a concentration of 1 : 500.</div>
			</div>
			</td>
		</tr>
		<tr>
			<td>4&#8242;,6-Diamidino-2&#8242;-phenylindole dihydrochloride (DAPI)</td>
			<td>Sigma Aldrich</td>
			<td>D9542</td>
			<td>
			<div>
			<div>1: 600 in KPBS-T.</div>
			</div>
			</td>
		</tr>
		<tr>
			<td>streptavidin- Cy3</td>
			<td>Thermo Fisher Scientific</td>
			<td>434315</td>
			<td>1:5000, rabbit.</td>
		</tr>
		<tr>
			<td>Anti-GAD65/67</td>
			<td>Abcam</td>
			<td></td>
			<td>1:2000, mouse.</td>
		</tr>
		<tr>
			<td>Anti-PV</td>
			<td>Sigma</td>
			<td>P3088</td>
			<td>1:200, goat.</td>
		</tr>
		<tr>
			<td>Anti-Chat</td>
			<td>Merk</td>
			<td>AB143</td>
			<td>1:5000, rabbit.</td>
		</tr>
		<tr>
			<td>Anti-NPY</td>
			<td>Immunostar</td>
			<td>22940</td>
			<td></td>
		</tr>
		<tr>
			<td>K<sub>2</sub>HPO<sub>4</sub></td>
			<td>Sigma-Aldrich</td>
			<td>P3786</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Sigma-Aldrich</td>
			<td>S3014</td>
			<td></td>
		</tr>
		<tr>
			<td>KH<sub>2</sub>PO<sub>4</sub></td>
			<td>Sigma-Aldrich</td>
			<td>NIST200B</td>
			<td>1:1000, chicken.</td>
		</tr>
		<tr>
			<td>Anti-GFP</td>
			<td>Abcam</td>
			<td>ab13970</td>
			<td></td>
		</tr>
		<tr>
			<td>Mounting solution</td>
			<td>Merck</td>
			<td>10981</td>
			<td>
			<div>
			<div>Polyvinyl alcohol mounting medium with DABCO&#174;, antifading</div>
			</div>
			</td>
		</tr>
		<tr>
			<td>OCT</td>
			<td>Agar Scientific</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Glycerol</td>
			<td>Sigma-Aldrich</td>
			<td>G9012-100ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Distilled water</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-freeze solution</td>
			<td>Tissue Pro Technology</td>
			<td>AFS05-1000N, 1000 mL/ea.</td>
			<td></td>
		</tr>
		<tr>
			<td><strong>EQUIPMENT FOR IMMUNOHISTOCHEMISTRY</strong></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Inverted fluorescence microscope</td>
			<td>Leica</td>
			<td>DMI6000 B</td>
			<td></td>
		</tr>
		<tr>
			<td>Confocal microscope</td>
			<td>Leica</td>
			<td>TCS SP8 laser scanning confocal microscope.</td>
			<td></td>
		</tr>
		<tr>
			<td>Prism</td>
			<td>GraphPad</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Microtome</td>
			<td>Leica</td>
			<td>SM2010R</td>
			<td></td>
		</tr>
	</tbody>
</table>

in vivo direct reprogramming of resident glial cells into interneurons by intracerebral injection of viral vectors

<p class="jove_content">将大脑中的常驻胶质体转化为体内功能性和亚型特异性神经元,为开发替代细胞替代疗法提供了一步,同时也创造了研究原位细胞命运的工具。迄今为止,通过体内重新编程获得神经元是可能的,但是这些神经元的精确表型或它们是如何成熟的,还没有得到详细的分析。在此协议中,我们使用基于 AAV 的病毒载体系统描述了对体内重新编程神经元的更有效的转换和细胞特异性识别。我们还为重新编程的细胞神经元成熟功能评估提供了一个协议。通过注射翻转切除(FLEX)载体,将重新编程和突生驱动报告基因注入大脑中作为细胞重新编程目标的特定细胞类型。这种技术允许轻松识别新重新编程的神经元。结果表明,获得重新编程的神经元功能随时间而成熟,接受突触接触,并显示出不同类型内神经元的电生理特性。使用转录因子Ascl1、Lmx1a和Nurr1,大多数重新编程的细胞具有快速窥探、含帕弗巴平的内神经元特性。</p>

<p class="jove_content" fo:keep-together.within-page="1">AAV载体的注入用于成功地将常驻NG2胶质细胞重新编程到NG2-Cre小鼠纹状体中的神经元中(<strong class="xfig">图1A)。</strong>为了专门针对NG2胶质,FLEX载体与重新编程/报告基因,插入在反义的方向,并两侧由两对反平行,异质性loxP位点(<strong class="xfig">图1B)。</strong>三个重编程基因(Ascl1、Lmx1a和Nurr1)中的每一个都置于单个载体上无处不在的cba启动子的控制之下。为了确保GFP表达仅限于来自Cre-表达细胞的重新编程的神经元,GFP被置于神经元特异性突触素启动子(也在FLEX向量中)的控制之下。</p>
<p class="jove_content" fo:keep-together.within-page="1">使用重新编程和报告器构造的组合允许在小鼠纹状体中生成 GFP 阳性神经元(<strong class="xfig">图 1C</strong><strong>,C'</strong>)。在没有重新编程基因的情况下使用报告器构造不会产生GFP阳性神经元(<strong class="xfig">图1D)。</strong></p>
<p class="jove_content" fo:keep-together.within-page="1">重新编程的神经元,是生物细胞内填充是可见的死后免疫染色(<strong class="xfig">图2A)。</strong>如果转换成功,应该有广泛的神经元形态。重新编程神经元的电生理记录显示存在与自发活动测量的午睡功能连接(<strong class="xfig">图2B,C)。</strong><strong></strong>这可以通过异位性GABAergic或谷氨酸阻滞剂(皮罗毒素或CNQX)来阻止,建议对重新编程的神经元进行兴奋和抑制性突触输入。自发活动的发生随着病毒注射后的时间而增加(<strong class="xfig">图2D),</strong>表明逐渐成熟。</p>
<p class="jove_content" fo:keep-together.within-page="1">电流诱导作用电位存在于功能神经元中。转换后,操作电位会随时间而增加 (<strong class="xfig">图 2E</strong>)。这进一步表明神经元功能的成熟。在未成熟的神经元中,电流将诱发无或非常少的动作电位(<strong class="xfig">图2F)。</strong></p>
<p class="jove_content" fo:keep-together.within-page="1">神经元的发射模式是细胞类型特定的,因为它取决于诸如细胞的形态和通道<sup class="xref">表达15</sup>等因素。体内重新编程的神经元中记录的模式可以区分为各组,与内源性神经元亚型(例如快速尖峰内神经元(细胞类型B,<strong class="xfig">图3B)</strong>或其他细胞类型(图3A,C,D)进行比较<strong class="xfig"></strong><strong></strong>).观察到的电生理差异可以通过存在特定的子类型标记和与GFP共表达(图3E-H)来<strong class="xfig"></strong><strong></strong>证实。总体而言,该数据表明,纹状体中存在的重新编程的神经元具有不同类型的内神经元的特性,如帕夫巴宾、ChAt-和NPY-表达的内神经元,以及纹状中尖质神经元标识(DARPP32+) (<strong class="xfig">图 3E</strong><strong>-H</strong>)</p>
<p class="jove_content" fo:keep-together.within-page="1"><img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/59465/59465fig1.jpg"><br><strong>图1:在体内重新编程驻地NG2胶质到神经元。</strong>(<strong>A</strong>) AAV病毒介导在体内重新编程的纹状NG2胶质的架构表示。(<strong>B</strong>) 用于体内重新编程的AAV5 FLEX结构的原理表示,其中基因表达由靶中细胞中的Cre表达调节。(<strong>C</strong>和<strong>C'</strong>) 体内重新编程的神经元,由 Syn-GFP + ALN 注射到 Striatum 中产生。(<strong>D</strong>) 没有重新编程的神经元,当没有重新编程的因素添加到病毒鸡尾酒,只有报告器构造注射在体内。刻度杆 = 100 毫米 (C),25 毫米(C'),25 毫米(D)。<a href="https://www.jove.com/files/ftp_upload/59465/59465fig1large.jpg" target="_blank">请点击此处查看此图的较大版本。</a></p>
<p class="jove_content" fo:keep-together.within-page="1"><img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/59465/59465fig2.jpg"><br><strong>图2:体内重新编程的神经元功能正常,并随着时间的推移显示成熟。</strong>(<strong>A</strong>) 生物素填充的重新编程神经元,显示成熟的神经元形态,包括树突状脊柱。痕迹显示 (<strong>B</strong>) 抑制 (GABAergic) 活性被阻断与皮洛毒素, GABA<sub>A</sub>受体拮抗剂和 (<strong>C</strong>) 兴奋活性被阻塞与 CNQX, AMPA 受体拮抗剂.(<strong>D</strong>) 具有午睡活动的神经元数量随着时间的推移而增加。(<strong>E</strong>) 修补神经元在注射后5周(w.p.i.)显示重复激发,并继续显示在8和12 w.p.i. (<strong>F</strong>) 未成熟神经元的电流诱导作用电位和发后活动,显示很少的突触事件和几个行动潜力相比 B 和 D. 比例尺 = 25 毫米<a href="https://www.jove.com/files/ftp_upload/59465/59465fig2large.jpg" target="_blank">请点击这里查看此图的较大版本。</a></p>
<p class="jove_content" fo:keep-together.within-page="1"><img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/59465/59465fig3v2.jpg"><br><strong>图3:体内重新编程的神经元显示纹状内神经的免疫性、化学和电生理特性。</strong>(<strong>A-D</strong>)体内重新编程神经元的发射模式可以是不同类型的:(A)A型类似于内源<strong></strong>性中性脊柱神经元(DARPP32+);(<strong>B</strong>) 类似于快发 (PV+) 内神经;(<strong>C</strong>) 类似于具有突出下垂 (NPY+) 的低阈值尖峰神经元;(<strong>D</strong>) 神经元以大后超极化 (Chat+) 激发。(<strong>E-H</strong>)共聚焦图像显示GFP和内神经元标记PV(E)、ChAT(F)、NPY(G)和投影神经元标记DARPP32(H)的共同定位。<strong></strong><strong></strong><strong></strong><strong></strong>所有刻度条 = 50 mm。<a href="https://www.jove.com/files/ftp_upload/59465/59465fig3v2large.jpg" target="_blank">请点击此处查看此图的较大版本。</a></p>

Watch this Scientific Journal Video about In Vivo Direct Reprogramming of Resident Glial Cells into Interneurons by Intracerebral Injection of Viral Vectors at JoVE.com

In Vivo Direct Reprogramming of Resident Glial Cells into Interneurons by Intracerebral Injection of Viral Vectors

在Vivo直接重新编程居民胶质细胞到内神经通过病毒载体的脑内注射

<p class="jove_content">该协议旨在利用大脑中基于AAV的病毒系统和FLEX突触素驱动的GFP报告器,在体内直接重新编程的内神经,从而在体内进行细胞识别和进一步分析。</p>

Converting resident glia in the brain into functional and subtype-specific neurons in vivo provides a step forward towards the development of alternative ...

<p>该协议表明，有可能直接从居民胶质产生新的神经元，这些重新编程的细胞成熟成亚型特异性神经元。主要优点是依赖 cre 的 AAV 病毒，它能够对 NG2-glia 和 GFP 报告器进行特定定位，该报告器标记重新编程的神经元进行分析。在大脑中产生新的神经元可能导致大脑细胞替代疗法的未来发展。</p><p>在我们的协议中，我们生成对精神疾病有影响的帕伐布明阳性内用神经。体内重新编程可以应用于其他大脑区域和电路，具体取决于您想要针对的神经元表型和神经系统状况。演示这个程序的将是珍妮·约翰逊：技术员，玛丽亚·佩雷拉：前博士生，马塞拉·比尔特尔：我们实验室的博士生。</p><p>为了产生AV5病毒载体，在5个T175烧瓶中用标准培养基播种 HEK293T 细胞。当细胞达到50-70%汇合时，准备以下混合进行转流。在50毫升离心管中，加入等量的矢量质粒和pDG系列帮助质粒。</p><p>将 Tris-EDTA 缓冲液添加到 144 微升的最终体积中，然后加入超纯水，使总体积达到 1296 微升并混合。接下来，加入144微升2.5摩尔氯化钙并混合。之后，立即将1.92毫升的HBS加入DNA溶液和涡流。</p><p>在室温下孵育60秒。随后，将溶液转移到28毫升的新鲜细胞培养中并混合。将烧瓶中的介质替换为含有转流混合物的介质。</p><p>等三天，把媒体转废了。在每个烧瓶中加入五毫升的收获缓冲液，然后在每个烧瓶中再添加四毫升的 DPBS，以冲洗剩余的细胞，并用细胞溶液池。将收获的细胞以1000xg离心，在4摄氏度下5分钟。</p><p>离心后，去除上流液，将颗粒溶解在15毫升的解解缓冲液中。将50毫升离心管冷冻在干冰上15分钟，并存放在零下20摄氏度的冰柜中。使用前，在37摄氏度的水浴中解冻收获的细胞。</p><p>在该过程中，通过碘沙醇梯度超离心进行 AAV 纯化，并在室温下使用离心度为 35 万 x g 的超离心吊管，使用 1 小时 45 分钟。要提取包含相的 AAV，请插入一个 10 毫升注射器，用 18 个量表针在 40 至 60% 相边界以下约 2 毫米，斜面朝上并退出。在提取了五到六毫升的病毒载体后，确保在到达蛋白质带之前停止。</p><p>然后，通过心安酸交换过滤器，通过不快于一滴的速度推动稀释的碘盐梯度，进行净化和浓缩。随后，缓慢地将三毫升的 IE 缓冲液通过过滤器进行清洗。接下来，将混合物用一到两毫升洗脱缓冲液将混合物倒入离心过滤单元中。</p><p>将 DPBS 添加到设备，最终体积为四毫升。在室温下以 2，000 x g 为单位的离心机，直到低于 0.5 毫升的 DPBS 留在过滤器中。之后，从管底取出液体，重新加注四毫升 DPBS，然后再次离心。</p><p>再重复此步骤两次，确保滤波器上的集中矢量体积在上次离心后约为 200 微升。使用移液器拆下 200 微升浓缩矢量，并通过 0.22 微米过滤器将其推至灭菌。接下来，将 200 微升等同物放入带联锁刀片的玻璃瓶中。</p><p>要注入重新编程因子，请将麻醉小鼠放在立体轴框架中。在手术开始时进行适当的镇痛，然后将注射针的玻璃毛细管的尖端放在呼吸管的正上方。确保毛细管尖端在 A-P 和 M-L 平面上完全直。</p><p>在数字坐标计数器上将 M-L 和 A-P 值重置为 0.0。要确保动物的头部处于完全平坦的位置，当 A-P 臂位于正 2.0 和减 2.0 以及 M-L 臂位于正 2.0 和减 2.0 时，使用数字坐标计数器测量 D-V 坐标值。相应地调整齿杆和耳杆的高度。</p><p>之后，稍微抬起注射器，在注射坐标处使用牙科钻孔钻孔。开始在现场钻孔，以循环和温和的方式工作。然后，将一块棉纱布放在打开的切口上，然后用盐水溶液冲洗注射器。</p><p>冲洗后，绘制一个气泡，然后一个微升溶液含有病毒载体。确保病毒溶液在气泡下方易于可视化。接下来，降低注射器，缓慢地向所需深度前进，并确保轨迹远离骨骼碎片。</p><p>随后，以每分钟0.4微升的速度注射一微升病毒溶液。注射完成后，在注射器提取前留出两分钟进行扩散。扩散后，慢慢缩回注射器，直到毛细管的尖端完全从大脑中撤出，然后小心缝合切口，将动物从立体气框架中取下。</p><p>在术后站中监测动物，直到恢复知觉。动物在病毒注射后被保存长达12周，以允许居民胶质重新编程成成熟的神经元。为了使用振动器为电生理学准备脑片，请将大脑从最旋转的部分分到高速的石质水平。</p><p>然后以275微米的速度以0.1毫米/秒的速度将花段分段。每个节后，小心地去除非注射的石室侧，将注射侧转移到小瓶中，并用底网和氧化 CREB 感应在室温下在水浴中滑动。将小瓶保持室温，直到所有部分被切割。</p><p>之后，慢慢将水浴的温度升高到37摄氏度，并离开30分钟，然后关闭加热器，让它冷却到室温。将一个组织部分转移到电生理学记录室后，将玻璃移液器安装在记录电极上，然后将其降低至溶液中。仔细检查电极的电阻。</p><p>然后，使用移液器缓慢接近重新编程的细胞，在电极中保持轻微的正压，以避免堵塞尖端，并在修补前检查该电池的 GFP 阳性。修补电池时，将电流夹的细胞从负 60 到负 70 毫伏，并注入 500 毫秒电流，从负 20 注入到正 90 皮奥安培，并注入 10 皮奥安佩雷增量以诱导作用电位。这表明神经元成熟和成功的重新编程。</p><p>随后，切换到电压夹并测量向内钠，并在 10 毫伏的降极步下延迟校正钾电流。这里是一个后记录生物细胞素填充重新编程神经元，显示成熟的神经元形态和树突状脊柱。在这里，重新编程的神经元的电生理记录显示，与自发活动措施存在术后功能连接。</p><p>跟踪显示抑制活性被阻断与皮罗毒素， GABAA受体拮抗剂和兴奋活性被阻塞与CNQX， AMPA受体拮抗剂.修补的神经元在注射后五周已经显示注射后活动，注射后8周和12周继续。具有当前诱导作用潜力的神经元数量也会随着时间的推移而增加。</p><p>更详细的分析揭示了几种不同的发射模式，其中大多数细胞表现出类似于帕瓦尔布明内维龙的快速尖峰活动。免疫石化学分析在12周进一步显示共同表达记者GP和帕瓦尔布明。按照此过程，您可以使用贴片查找技术检查基因表达，或评估具有单突触追踪和 iDISCO 的三维突触连接。</p><p>现在，有可能重新编程居民胶质到帕瓦尔布明表达实习，我们已经开始调查这些是否是真实的，可以用作治疗工具。在处理动物和使用 AAV 病毒时，请记住遵循既定的协议和准则。</p>

Research

JoVE Journal

Neuroscience

Intracranial Injection of Adeno-associated Viral Vectors

颅内注射腺相关病毒载体

Intracranial injection of viral vectors engineered to express a fluorescent protein is a versatile labeling technique for visualization of specific ...

科研

神经科学

Autologous Blood Injection to Model Spontaneous Intracerebral Hemorrhage in Mice

自体血液的注入模型小鼠自发性脑出血

Investigation of the pathophysiology of injury after intracerebral
hemorrhage (ICH) requires a reproducible animal model.  While ICH
accounts for 10-15% ...

Transfection of Mouse Retinal Ganglion Cells by <em>in vivo</em> Electroporation

Transfection of Mouse Retinal Ganglion Cells by in vivo Electroporation

小鼠视网膜神经节细胞的转染<em>在体内</em>电穿孔

The targeting and refinement of RGC projections to the midbrain is a popular and powerful model system for studying how precise patterns of neural ...

Expansion of Embryonic and Adult Neural Stem Cells by <em>In Utero</em> Electroporation or Viral Stereotaxic Injection

Expansion of Embryonic and Adult Neural Stem Cells by In Utero Electroporation or Viral Stereotaxic Injection

扩展胚胎及成体神经干细胞<em&gt;在子宫内</em电穿孔或病毒立体定向注射

Somatic stem cells can divide to generate additional stem cells (expansion) or more differentiated cell types (differentiation), which is fundamental for ...

Lineage-reprogramming of Pericyte-derived Cells of the Adult Human Brain into Induced Neurons

谱系重编程的成人大脑的周细胞源性细胞诱导成神经细胞

Direct lineage-reprogramming of non-neuronal cells into induced neurons (iNs) may provide insights into the molecular mechanisms underlying neurogenesis ...

Inducing Plasticity of Astrocytic Receptors by Manipulation of Neuronal Firing Rates

诱导星形胶质细胞受体的可塑性由神经元的放电频率操作

Close to two decades of research has established that astrocytes in situ and in vivo express numerous G protein-coupled receptors (GPCRs) that can be ...

Limbal Approach-Subretinal Injection of Viral Vectors for Gene Therapy in Mice Retinal Pigment Epithelium

病毒载体的基因治疗的小鼠视网膜色素上皮缘方针，视网膜下注射

The eye is a small and enclosed organ which makes it an ideal target for gene therapy. Recently various strategies have been applied to gene therapy in ...

Intracerebroventricular and Intravascular Injection of Viral Particles and Fluorescent Microbeads into the Neonatal Brain

病毒颗粒和荧光微球侧脑室和血管内注入新生儿脑

In the study on the pathogenesis of viral encephalitis, the infection method is critical. The first of the two main infectious routes to the brain is the ...

Cannula Implantation into the Cisterna Magna of Rodents

将套管植入啮齿动物的麦格纳池中

Cisterna magna cannulation (CMc) is a straightforward procedure that enables direct access to the cerebrospinal fluid (CSF) without operative damage to ...

Rat Model of Widespread Cerebral Cortical Demyelination Induced by an Intracerebral Injection of Pro-Inflammatory Cytokines

由亲炎细胞因子的内脑注射诱导的广度脑皮质脱叶的鼠模型

Multiple sclerosis (MS) is the most common immune-mediated disease of the central nervous system (CNS) and progressively leads to physical disability and ...