我们非常感谢中国国家自然科学基金（第31800903项，授予郑伟）和温州科学技术计划（第Y2020426号，授予翁耀彦）对这项工作的支持。

本研究中描述的所有动物程序均按照美国国立卫生研究院《实验动物护理和使用指南》中概述的指南进行，并经温州医科大学机构动物护理和使用委员会批准。
1. 动物
<ol><li>购买年龄为8-12周，体重为20-22g的雄性C57BL / 6小鼠。</li>
	<li>获得转基因Rosa-LSL（Lox-StoP-Lox）-tdTomato（Ai9）小鼠系和雄性A2AR flox/ flox 小鼠。</li>
	<li>将小鼠随机分配到两组并饲养在笼子中，每个笼子最多5只小鼠，在标准的12小时光照/ 12小时黑暗循环下1周。</li>
	<li>为小鼠提供足够的食物和水，并将它们保持在25°C的恒定温度下。</li>
</ol>2. 用 AAV2/5-shRNA 特异性敲低 A2A R
<ol><li>称量每只鼠标的重量并记下值。</li>
	<li>用1%戊巴比妥钠腹膜内麻醉小鼠，剂量为6-8mL / kg，相当于60-80mg / kg戊巴比妥，并将小鼠放在加热垫上保温。 注意：根据小鼠的体重给予正确剂量的戊巴比妥钠以确保手术成功至关重要。麻醉不宜太深，因为它可能导致死亡，但不应太浅，因为小鼠可能会在手术过程中醒来，从而可能影响病毒注射的有效性。通过捏住脚趾并确保小鼠没有反应来检查适当的麻醉剂量。</li>
	<li>使用专门的剃须刀修剪麻醉小鼠的顶毛。</li>
	<li>在双眼上涂抹眼药膏以防止干燥，并将小鼠固定在立体定位装置上以固定大脑。用无菌窗帘盖住动物。</li>
	<li>用三轮碘伏消毒液和75%酒精对小鼠头颈部皮肤进行彻底消毒，以减少术后感染。然后局部将1%利多卡因局部施加在小鼠的头皮上，并做一个小切口，在显微镜下完全暴露颅骨。 注意：在整个过程中使用无菌器械。</li>
	<li>准备两个 10 μL 注射器：一个装有购买的 AAV2/5-A 2A R-shRNA（滴度：6.27 × 10 9 vg/μL），另一个装有购买的 AAV2/5-Scramble（滴度：6.21× 109 vg/μL）。 注：使用注射器时，前端需要连接玻璃毛细管，通过控制玻璃毛细管的长度可以获得10μL的范围。</li>
	<li>使用显微镜定位前囟和λ并设置坐标点（AP：-0.58;毫升：±1.10;DL：-2.20）在10μL注射器上（图1）。该坐标点基于小鼠大脑立体定位图谱15。</li>
	<li>在调整后的坐标点在头骨上钻一个小孔。 注意：在显微镜下，使用无菌微钻对头骨表面进行钻孔。钻孔后，通过去除覆盖大脑的脑膜并暴露下面的脑实质，可以防止血管损伤。这一步可以防止玻璃毛细管的尖端被脑膜折断。钻孔的直径应足以使针尖进入脑组织。</li>
	<li>通过侧向注射以 100 nL/min 的稳定速率将 2 μL AAV2/5-A2AR-shRNA 病毒注入脑室。在拔出之前将针头保持在原位 10 分钟。请注意，该病毒是通过单侧注射给药的。</li>
	<li>立即使用医用生物纤维蛋白胶水密封受伤的皮肤，以防止病毒流失，如果不快速涂抹胶水，可以在取下针头后用脑脊液冲洗干净。此外，不要使用棉签擦拭脑脊液，因为它也可能导致病毒丢失。</li>
	<li>为了促进恢复，将鼠标放在加热垫上，将体温保持在37.0±0.5°C，并将1%利多卡因局部涂抹在小鼠的头皮上。在诱导EAE模型之前，让小鼠休养2周，因为这段时间是AAV2 / 5-A 2A R-shRNA病毒表达和随后的A2AR敲低所必需的。</li>
</ol>3. 具有 X-overP1 Cre/位点 （Cre/LoxP） 系统的 A 2A R 的 ChP 特异性敲低
注意：可以使用前面描述的方法实现以下过程。有关详细的注射方法，请参阅步骤 2.1-2.11。
<ol><li>将 2 μL CRE-TAT 重组酶注射到 Rosa-LSL （Lox-StoP-Lox）-tdTomato 小鼠的每个侧脑室中作为实验组，将 2 μL 无菌磷酸盐缓冲盐水 （PBS） 作为对照组。使用与上述相同的方案（第2节），将2μL的CRE-TAT重组酶注射到A2AR flox/ flox 小鼠的每个侧脑室中，以及2μL无菌PBS作为对照组。</li>
	<li>准备冷冻组织切片并在注射 CRE-TAT 重组酶后 2 周进行核染色。有关详细信息，请参阅步骤 4.1-4.3。</li>
</ol>4.小鼠心内灌注
<ol><li>施用60-80mg / kg戊巴比妥钠对小鼠进行深度麻醉。通过捏脚趾确认麻醉平面，并使用 40 mL 无菌 PBS 溶液和 20 mL 4% 多聚甲醛 （PFA） 进行经心灌注。 注意：肝脏中的白色表明灌注成功，而灌注过程中肺肿大或PBS从口腔流出表明手术失败。</li>
	<li>快速提取小鼠大脑，确保最小的蛋白质降解。</li>
	<li>将小鼠脑浸没在4%PFA / PBS中过夜用于固定后，然后用30%蔗糖PB溶液替换72小时。 注意：避免脑组织过度脱水至关重要，因此大脑不应在蔗糖PB溶液中停留太久。</li>
</ol>5. 冷冻组织切片和染色
<ol><li>将先前脱水的小鼠大脑嵌入最佳切割温度（OCT）胶中，冷冻嵌入的大脑，并使用滑动切片机切割厚度为20μm的冠状切片。</li>
	<li>将大脑部分放在载玻片上。一旦脑切片干燥，将它们储存在-20°C冰箱中用于后续实验。</li>
	<li>将每个载玻片放入框架盒中，然后将框架浸入装有 PBS 的容器中以彻底冲洗载玻片。用PBS溶液轻轻冲洗脑载玻片三次，每次10分钟。 注意： 应小心，以防止大脑部分从载玻片上掉下来。</li>
	<li>用4′，6-二脒基-2-苯基吲哚（DAPI）溶液染色脑载玻片10分钟。</li>
	<li>用PBS溶液冲洗脑载玻片5分钟。</li>
	<li>将一滴抗淬灭封固剂涂在大脑部分。</li>
	<li>将盖玻片放在脑切片上，用指甲油密封盖玻片，并使用常规荧光显微镜分析切片。</li>
</ol>6. EAE诱导
注意：在 shRNA 或 CRE-TAT 重组酶注射 2 周后进行 EAE 诱导11.
<ol><li>通过将 2.5 mg 髓鞘少突胶质细胞糖蛋白 （MOG35-55） 与 2 mL PBS 混合来制备水溶液。通过将 结核分枝杆菌 （H37Ra） 与不完全弗氏佐剂 （IFA） 混合来制备完整的弗氏佐剂 （CFA） 油溶液。</li>
	<li>将水溶液和油溶液以 1：1 的比例混合。使用三通管将混合物搅打到水包油状态。 注意： 三通管也称为三通管。它是一种塑料管，可以控制溶液的流动方向，使MOG35-55 和CFA充分混合。</li>
	<li>使用高速均质机在冰浴条件下为EAE模型制作MOG抗原乳液。</li>
	<li>用1%戊巴比妥钠腹膜内麻醉小鼠，剂量为6-8mL / kg，相当于60-80mg / kg戊巴比妥。</li>
	<li>皮下注射MOG抗原乳剂，体积为10mL / kg，每次注射四次。 注意：仔细选择注射部位很重要，因为不同的部位可能对小鼠的发病率和死亡率产生不同的影响。此外，重复注射MOG会导致免疫耐受，因此研究小组选择了一种单一注射方法来防止这种潜在问题。</li>
	<li>注射 MOG 后立即腹膜内注射 500 ng/mL 百日咳毒素 （PT），剂量为 5 mg/kg。 注意：PT增加血脑屏障（BBB）的通透性，并促进T细胞浸润到大脑中。</li>
	<li>48小时后，腹膜内以相同体积注射PT溶液。</li>
</ol>7. 神经功能缺损评分
<ol><li>每天使用从0到15的评定量表16 对小鼠进行评估和分级，以根据以下神经功能缺损评估EAE的发生率和严重程度： 尾巴：0 表示没有迹象，1 表示半瘫痪的尾巴，2 表示完全瘫痪的尾巴。 四肢：0 表示没有体征，1 表示步态无力或改变，2 表示瘫痪，3 表示肢体完全瘫痪。</li>
	<li>给完全瘫痪的四肢瘫痪动物打 12 分，给死亡率打 15 分。</li>
</ol>8. 苏木精-伊红（H&E）染色
<ol><li>取脱水的小鼠大脑并将其嵌入熔融石蜡中。让石蜡块冷却并固化以备后用。 注意：石蜡块应完全干燥和冷却，以避免组织损伤。</li>
	<li>将小鼠脑石蜡块切成5μm厚的载玻片。将石蜡小鼠脑切片放在载玻片上，并在60°C烘箱中干燥3小时。</li>
	<li>将载玻片依次浸入二甲苯溶液I中10分钟，二甲苯溶液II浸泡10分钟，100%酒精I浸泡3分钟，100%酒精II浸泡3分钟，95%酒精浸泡3分钟，90%酒精浸泡3分钟，80%酒精浸泡3分钟，浸泡在蒸馏水中1分钟。</li>
</ol>9. 定量聚合酶链反应（qPCR）分析
<ol><li>用PBS灌注小鼠后，从心室中取出ChP并用Trizol提取RNA。使用第一个链 cDNA 合成试剂盒合成 cDNA。</li>
	<li>使用Ex Taq SYBR-green预混液和实时荧光定量PCR系统进行qPCR分析。使用以下 A2AR 引物：forward - GCCATCCCATTCGCCATCA;反向 - GCAATAGCCAAGAGGCTGAAGA。</li>
</ol>

脉络丛中的选择性基因沉默

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提交人表示，他们没有相互竞争的经济利益或其他披露要申报。

该研究提出了两种不同的方法来靶向敲低ChP基因。第一种方法涉及将含有Cre重组酶的CRE-TAT注射到A2AR flox/ flox小鼠中。第二种方法需要ICV注射携带A2AR的shRNA的AAV2/5。通过利用这两种策略，本工作实现了 ChP 中 A 2A R 的选择性敲低，并能够证明抑制 ChP 中 A2AR 信号传导对 EAE 病理学的保护作用。值得注意的是，该协议包括两个关键步骤。首先，立体定位操作是必...

脉络丛 （ChP） 通常被认为通过分泌脑脊液 （CSF） 和脑源性神经营养因子 （BDNF） 来帮助维持脑功能稳态1,2。在过去三十年中，越来越多的研究表明，ChP 代表了免疫细胞浸润到中枢神经系统 （CNS） 的独特途径。
ChP 的紧密连接 （TJ） 由单层 ChP 上皮细胞组成，通过阻止大分子和免疫细胞进入大脑来维持免疫稳态3。然而，在某些病理条件下，ChP 组织会检测脑脊液和血液中的危险相关分子模式 （DAMP） 并做出反应，导致异常免疫浸润和脑功能障碍 4,5。尽管ChP起着关键作用，但它的小尺寸和在大脑中的独特位置使得很难在不影响其他大脑区域的情况下研究其功能。因此，在ChP中特异性地操纵基因表达是了解其功能的理想方法。
最初，环化重组酶 （Cre） 转基因系在 ChP 中表达的基因特异性启动子的控制下表达 Cre，通常用于通过与 flox 候选基因杂交来删除靶基因 6,7,8。例如，转录因子叉头盒J1（FoxJ1）仅在产前小鼠脑的ChP上皮细胞中表达7。因此，FoxJ1-Cre 系通常用于删除位于 ChP 6,9 中的基因。然而，这种策略的成功很大程度上取决于启动子的特殊性。逐渐发现 FoxJ1 的表达模式不够独特，因为 FoxJ1 也存在于大脑和外周系统其他部位的纤毛上皮细胞中7。为了克服这一局限性，进行了脑室内 （ICV） 注射 Cre 重组酶，以将重组酶输送到絮状转基因系的脑室中。该策略显示出高度特异性，仅在 ChP 组织中存在 tdTomato 荧光就证明了这一点 10,11。然而，这种方法仍然受到氟化转基因小鼠品系可用性的限制。为了解决这个问题，研究人员采用了腺相关病毒 （AAV） 的 ICV 注射来实现 ChP 特异性敲低或靶基因的过表达12,13。对不同 AAV 血清型的 ChP 感染的综合评估显示，AAV2/5 和 AAV2/8 在 ChP 中表现出很强的感染能力，而不感染其他大脑区域。然而，发现 AAV2/8 感染心室周围的室管膜，而 AAV2/5 组未显示感染14。该方法的优点是克服了获得floxed转基因动物的局限性。
本文描述了使用两种方法在 ChP 中敲低基因的分步方案：携带腺苷 A 2A 受体 （A 2A R） 的 shRNA 的 AAV2/5 的 ICV 和由 TAT 序列 （CRE-TAT） 重组酶组成的 Cre 重组酶蛋白，以实现 A2A R 的 ChP 特异性敲低。研究结果表明，敲低 ChP 中的 A2AR 可以缓解实验性自身免疫性脑脊髓炎 （EAE）。该详细方案为 ChP 功能研究和 ChP 中基因的特异性敲低提供了有用的指导。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>A2ARflox/flox mice</td>
			<td>State Key Laboratory of Ophthalmology, Optometry and Visual Science, Wenzhou Medical University</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>AAV2/5-A2AR-ShRNA virus</td>
			<td>Shanghai Heyuan Biotechnology Co. LTD</td>
			<td>pt-4828</td>
			<td></td>
		</tr>
		<tr>
			<td>antifade mounting medium</td>
			<td>Beyotime Biotechnology</td>
			<td>0100-01</td>
			<td></td>
		</tr>
		<tr>
			<td>borosilicate glass capillary</td>
			<td>Beijing Meiyaxian Technology Co. Ltd</td>
			<td>B100-50-10</td>
			<td></td>
		</tr>
		<tr>
			<td>brain stereotaxic apparatus</td>
			<td>RWD, Shenzhen</td>
			<td>69100</td>
			<td></td>
		</tr>
		<tr>
			<td>C57BL/6 mice</td>
			<td>Beijing Vital Charles River Laboratory Animal Technology Company</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>CRE-TAT recombinase</td>
			<td>Millipore</td>
			<td>SCR508</td>
			<td></td>
		</tr>
		<tr>
			<td>DAPI</td>
			<td>Absin</td>
			<td>B25A031</td>
			<td></td>
		</tr>
		<tr>
			<td>frozen slicing machine</td>
			<td>Leica</td>
			<td>CM1950</td>
			<td></td>
		</tr>
		<tr>
			<td>H37Ra</td>
			<td>Becton Dickinson and company</td>
			<td>231141</td>
			<td></td>
		</tr>
		<tr>
			<td>Hamilton syringe</td>
			<td>Hamilton, American</td>
			<td>P/N:&#160;86259</td>
			<td></td>
		</tr>
		<tr>
			<td>Incomplete Freunds adjuvant</td>
			<td>Sigma</td>
			<td>F5506</td>
			<td></td>
		</tr>
		<tr>
			<td>Laser confocal microscope</td>
			<td>Zeiss</td>
			<td>LSM900</td>
			<td></td>
		</tr>
		<tr>
			<td>MOG35-55</td>
			<td>Suzhou Qiangyao Biotechnology Co., LTD</td>
			<td>4010006243</td>
			<td></td>
		</tr>
		<tr>
			<td>OCT glue</td>
			<td>Epredia</td>
			<td>6502p</td>
			<td></td>
		</tr>
		<tr>
			<td>paraformaldehyde</td>
			<td>Chengdu Kelong Chemical Reagent Company</td>
			<td>30525-89-4</td>
			<td></td>
		</tr>
		<tr>
			<td>pentobarbital sodium</td>
			<td>Boyun Biotech</td>
			<td>PC13003</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipette gun</td>
			<td>Eppendorf</td>
			<td>N45014F</td>
			<td></td>
		</tr>
		<tr>
			<td>PrimeScript 1st Strand cDNA Synthesis Kit</td>
			<td>Takara</td>
			<td>&#160;6110A</td>
			<td></td>
		</tr>
		<tr>
			<td>Real- Time PCR System</td>
			<td>BioRad</td>
			<td>CFX96</td>
			<td></td>
		</tr>
		<tr>
			<td>Rosa-LSL (Lox-StoP-Lox)-tdTomato mice</td>
			<td>Jackson Laboratory</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>sucrose</td>
			<td>Sangon Biotech</td>
			<td>A502792-0500</td>
			<td></td>
		</tr>
		<tr>
			<td>super high speed homogenizer</td>
			<td>IKA</td>
			<td>3737025</td>
			<td></td>
		</tr>
		<tr>
			<td>Trizol</td>
			<td>Invitrogen</td>
			<td>15596026</td>
			<td></td>
		</tr>
		<tr>
			<td>xylene solution</td>
			<td>Chengdu Kelong Chemical Reagent Company</td>
			<td>1330-20-7</td>
			<td></td>
		</tr>
	</tbody>
</table>

targeted knockdown of genes in the choroid plexus

脉络丛 （ChP） 是免疫细胞在生理和病理条件下浸润到中枢神经系统 （CNS） 的关键通道。最近的研究表明，调节 ChP 活性可以预防中枢神经系统疾病。然而，由于其微妙的结构，在不影响其他大脑区域的情况下研究 ChP 的生物学功能具有挑战性。本研究提出了一种使用腺相关病毒 （AAV） 或由 TAT 序列组成的环化重组酶 （Cre） 重组酶 （CRE-TAT） 重组酶蛋白在 ChP 组织中敲低基因的新方法。结果表明，将AAV或CRE-TAT注射到侧脑室后，荧光完全集中在ChP中。 利用这种方法，该研究利用RNA干扰（RNAi）或X-overP1（Cre/LoxP）系统的Cre/位点成功敲低了ChP中的腺苷A 2A受体（A2A R），并表明这种敲低可以缓解实验性自身免疫性脑脊髓炎（EAE）的病理。该技术可能对未来研究 ChP 在中枢神经系统疾病中的作用具有重要意义。

The choroid plexus (ChP) was often thought to help maintain brain functional homeostasis by secreting cerebrospinal fluid (CSF) and brain-derived neurotrophic factor (BDNF)1,2. Increasing research over the last three decades has revealed that the ChP represents a distinct pathway for immune cell infiltration into the central nervous system (CNS).
The tight junctions (TJs) of the ChP, composed of a monolayer ChP epithelium, maintain immunological homeostasis by preventing macromolecules and immune cells from entering the brain3. However, under certain pathological conditions, the ChP tissue detects and responds to danger-associated molecular patterns (DAMPs) in the CSF and blood, leading to abnormal immune infiltration and brain dysfunction4,5. Despite its critical role, the ChP&#39;s small size and unique location in the brain make it difficult to study its function without affecting other brain regions. Therefore, manipulating gene expression specifically in the ChP is an ideal approach to understanding its function.
Initially, cyclization recombination enzyme (Cre) transgenic lines, which express Cre under the control of promoters specific to genes expressed in the ChP, were commonly used to delete target genes by breeding with floxed candidate genes6,7,8. For example, the transcription factor Forkhead box J1 (FoxJ1) is exclusively expressed in the ChP epithelium of the prenatal mouse brain7. Thus, the FoxJ1-Cre line was often used to delete genes located in the ChP6,9. However, the success of this strategy relies heavily on the specificity of the promoter. It was gradually discovered that the FoxJ1 expression pattern was not distinctive enough, as FoxJ1 was also present in ciliated epithelial cells in other parts of the brain and peripheral system7. To overcome this limitation, intra-cerebroventricular (ICV) injection of Cre recombinase was performed to deliver recombinase into the ventricles of floxed transgenic lines. This strategy showed high specificity, as evidenced by the presence of tdTomato fluorescence solely in the ChP tissue10,11. However, this method is still limited by the availability of floxed transgenic mouse lines. To address this issue, researchers have employed ICV injection of adeno-associated virus (AAV) to achieve ChP-specific knockdown or the overexpression of target genes12,13. A comprehensive evaluation of different AAV serotypes for ChP infection revealed that AAV2/5 and AAV2/8 exhibit strong infection abilities in the ChP, while not infecting other brain regions. However, AAV2/8 was found to infect the ependyma surrounding ventricles, whereas the AAV2/5 group showed no infection14. This method has the advantage of overcoming the limitations of acquiring floxed transgenic animals.
This article describes a step-by-step protocol for gene knockdown in the ChP using two methods: ICV of AAV2/5 carrying shRNA of the adenosine A2A receptor (A2AR) and Cre recombinase protein consisting of TAT sequence (CRE-TAT) recombinase to achieve ChP-specific knockdown of A2AR. The study findings suggest that knocking down A2AR in the ChP can alleviate experimental autoimmune encephalomyelitis (EAE). This detailed protocol provides useful guidance for ChP function studies and the specific knockdown of genes in the ChP.

作者聚焦：脉络丛研究中基因表达操作技术的见解和创新 

脊索丛中基因的靶向敲除

The objective of this research is to investigate the techniques for manipulating gene expression in the choroid plexus. Our study has introduced the two methods that allow for technique exclusively in the choroid plexus without any discernible impact on other regions. Our findings can potentially enhance future function studies of the choroid plexus in neuroscience.Our research has reviewed that inhibiting T-cell infiltration across the choroid plexus through selective lockdown of adenosine to a receptor is sufficient to protect mice from EAE pathology. This finding supports the notion that detecting gateway activity in the choroid plexus could present a promising new strategy for treating multiple sclerosis. This study presents two alternative methods for gene expression manipulation in the choroid plexus.ICV injection of Cre recombinase into those transgenic mice or ICV injection of AAV2/5, or it's targeted SHI in the WT mice. These methods overcome issues with promoting specificity and transgenetic mouse breeding.

通过 ICV 注射 AAV2/5-shRNA 或 CRE-TAT 敲低 ChP 特异性 A2AR A2AR 在 ChP 中作为 EAE 发病机制中神经信息的强大调节因子的作用尚不清楚。敲低 ChP 特异性 A 2A R 表达可以揭示 A2AR 对 EAE和其他神经系统炎症中枢免疫系统的调节作用。本研究使用 ICV 注射 CRE-TAT 来降低 A 2A R flox/flox 小鼠 ChP 中 A2AR 的表达。为了确保 ChP 特异性，我们首先将 ...

Watch this Scientific Journal Video about Insights and Innovations in Gene Expression Manipulation Techniques for Choroid Plexus Research at JoVE.com

在这里，我们描述了一种选择性地改变脉络丛中基因表达的方法，同时避免对其他大脑区域的任何影响。

The choroid plexus (ChP) serves as a critical gateway for immune cell infiltration into the central nervous system (CNS) under both physiological and ...

本研究的目的是研究操纵脉络丛中基因表达的技术。我们的研究引入了两种方法，这些方法只允许在脉络丛中使用技术，而不会对其他区域产生任何明显的影响。我们的研究结果可能会增强神经科学中脉络丛的未来功能研究。我们的研究回顾了通过选择性锁定腺苷到受体来抑制 T 细胞浸润脉络丛足以保护小鼠免受 EAE 病理学的影响。这一发现支持了这样一种观点，即检测脉络丛中的门户活动可能为治疗多发性硬化症提供一种有前途的新策略。本研究提出了两种在脉络丛中进行基因表达操作的替代方法。ICV 将 Cre 重组酶注射到这些转基因小鼠中或 ICV 注射 AAV2/5，或者在 WT 小鼠中靶向 SHI。这些方法克服了促进特异性和转基因小鼠育种的问题。

targeted-knockdown-of-genes-in-the-choroid-plexus

Research

JoVE Journal

Neuroscience

Targeted Knockdown of Genes in the Choroid Plexus

687 次观看.  Wenzhou Medical University. 本研究的目的是研究操纵脉络丛中基因表达的技术。我们的研究引入了两种方法，这些方法只允许在脉络丛中使用技术，而不会对其他区域产生任何明显的影响。我们的研究结果可能会增强神经科学中脉络丛的未来功能研究。我们的研究回顾了通过选择性锁定腺苷到受体来抑制 T 细胞浸润脉络丛足以保护小鼠免受 EAE 病理学的影响。这一发现支持了这样一种观点，即检?...

视频: 作者聚焦：脉络丛研究中基因表达操作技术的见解和创新 

The choroid plexus (ChP) serves as a critical gateway for immune cell infiltration into the central nervous system (CNS) under both physiological and pathological conditions. Recent research has shown that regulating ChP activity may offer protection against CNS disorders. However, studying the biological function of the ChP without affecting other brain regions is challenging due to its delicate structure. This study presents a novel method for gene knockdown in ChP tissue using adeno-associated viruses (AAVs) or cyclization recombination enzyme (Cre) recombinase protein consisting of TAT sequence (CRE-TAT). The results demonstrate that after injecting AAV or CRE-TAT into the lateral ventricle, the fluorescence was exclusively concentrated in the ChP. Using this approach, the study successfully knocked down the adenosine A2A receptor (A2AR) in the ChP using RNA interference (RNAi) or Cre/locus of X-overP1 (Cre/LoxP) systems, and showed that this knockdown could alleviate the pathology of experimental autoimmune encephalomyelitis (EAE). This technique may have important implications for future research on the ChP's role in CNS disorders.

Here, we describe a method to selectively alter gene expressions in the choroid plexus while avoiding any impact in other brain areas.

科研

神经科学

ChP Specific Knockdown of A2ARs with AAV2/5-shRNA and Cre/LoxP System

ChP Specific Knockdown of A2ARs with AAV2/5-shRNA and Cre/LoxP System  

To begin, place the anesthetized mice on the surgical platform. After trimming the hair, apply eye ointment down both eyes to prevent them from drying. Then fix the mice onto a stereotaxic apparatus for immobilization.Make a small incision under a microscope to fully expose the skull. Prepare two 10 microliter syringes, one containing AAV2/5-adenosine 2A receptor shRNA, and the other containing AAV2/5 scramble. Then use the microscope to locate the bregma and lambda and set the coordinate point on the 10 microliter syringe based on the mouse brain stereotaxic atlas.Drill a small hole in the skull at the established coordinate point. Inject two microliters of AAV2/5-Adenosine 2A receptor shRNA virus into the brain ventricle via lateral injection at a steady rate of 100 nanoliters per minute. Keep the needle in place for 10 minutes before withdrawing.Seal the injured skin promptly using medical bio fibrin glue to prevent virus loss. Place the mouse on a heating pad, maintaining its body temperature approximately at 37 degrees Celsius to facilitate recovery. Inject two microliters of CRE-TAT Recombinase into each lateral ventricle of Arosa L-D-L-T-D tomato mouse for the experimental group, and two microliters of sterile PBS for the control group.Two weeks after the CRE-TAT Recombinase injection, prepare frozen tissue sections, perform nuclear staining and prepare the slide for imaging Knocking down adenosine 2A receptor in the choroid plexus in homozygous adenosine 2A receptor fluxed mouse, using CRE-TAT, led to decreased EAE clinical scores. Similarly, knocking down choroid plexus in adenosine 2A receptor wild-type mice, using AAV2/5 shRNA decreased EAE clinical scores. The QPCR analysis showed that the mRNA levels of adenosine 2A receptor decreased in the AAV2/5 shRNA and CRE-TAT groups, compared to each control.Silencing adenosine 2A receptor, specifically in the choroid plexus, reduced immune cell infiltration into the spinal cord.