作者要感谢 NINDS 动物护理机构工作人员的支持。这项工作得到了 NIH 校内研究部 NINDS 的支持（年度报告编号 1ZIANS003129）。内容完全由作者负责，并不一定代表美国国立卫生研究院的官方观点。

临床前小鼠模型中通过静脉注射进行基于 AAV 的治疗

<ol>
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作者没有与本文中发表的工作相关的任何披露。

由于 AAV 作为基因治疗载体的多功能性，基于 AAV 的疗法对单基因疾病具有巨大潜力，这使得定制 AAV 以满足不同疾病的各种递送需求成为可能 4,5,7,9。在临床前小鼠模型中，AAV 通常通过静脉注射给药，以测试潜在疗法的安全性和有效性16。由于不同的 AAV 注射剂量会导致实验结果的显着差异，因此实验者能够始终如一地注射预期的 AAV 剂量以确保生成的体内数据的有效性和稳健性至关重要28。静脉注射被广泛使用，但它们在技术上具有挑战性，需要广泛的培训和持续的实践来发展和保持确保持续成功注射的技能水平 16,17,18,19。除了正确注射 AAV 外，通常还需要使用检测来评估注射的 AAV 向目标组织或细胞的生物分布和递送效率29,30。
该方案旨在通过全面描述在 7-9 周龄未镇静小鼠中管理 AAV 的优化 IV 注射方案的细节，帮助实验者轻松、成功和一致地进行 IV 注射。重要的是要注意，由于静脉可见度降低或与该方法中使用的限制器不相容，在此处使用的年龄范围内明显小于或大于野生型小鼠的小鼠可能会带来更大的挑战。先前已经报道，由于血管尺寸小，尾部静脉注射不适合在 6 周龄以下的小鼠中静脉注射试剂31。尽管有可能，但可能很难持续注射体重低于 22.0 克的小鼠。使用非典型大小小鼠的研究人员可能需要适应该程序。该方案还概述了可用于评估 AAV 生物分布和转导效率的几种测定方法。
在遵循此协议时，需要牢记一些关键点。在注射过程中，如果针头不在静脉内，则 29 G 针头会提供更大的阻力。这减少了注射尝试失败期间因意外血管周围注射溶液而损失的体积。胰岛素注射器的死体积比普通注射器小。如果使用与此处列出的注射器和/或针头不同的注射器和/或针头，则可能需要在方案步骤 1.1.3.3 中准备额外的注射液体积，以解决更大的死腔体积（例如，在预期剂量中加入 30 μL 而不是 15 μL）。
如果在将 AAV 剂量吸入注射器时在注射器侧面形成细小的吸入引起的气泡，请慢慢地将注射液进一步拉到注射器上。这将去除大多数小气泡。将至少 10-15 μL AAV 额外加载到要注射的预期体积中。此额外体积用于说明在排出气泡或可能失败的进样尝试期间可能丢失的任何体积。（例如，如果要注射的目标体积为 150 μL，则将 165 μL 加载到注射器中（注射器刻度上 160 μL 和 170 μL 标记之间的中间位置）。如果针头正确放置在静脉内，并且在成功进样尝试前注射器中的体积为 165 μL，则输送试剂直到注射器中剩余 15 μL（介于 10 μL 和 20 μL 标记之间），从而输送 150 μL （165 μL - 150 μL = 15 μL））。将斜面管腔（斜面朝上）与注射器刻度对齐，可以在注射过程中跟踪输送的体积。
一些实验者可能更喜欢将鼠标侧放，这样与脚上的鼠标相比，它的一条静脉是笔直的且易于接近的。但是，在注射不同大小的小鼠时，根据鼠标的大小，小鼠侧面的尾巴会以不同的角度倾斜，需要调整注射角度。这可能会对手术成功的一致性产生负面影响。在最初的练习尝试中，实验者可以尝试两种鼠标约束方向，以确定他们喜欢的方法。让鼠标脚着可以快速轻松地进入两侧侧尾静脉。这减少了在多次注射尝试失败的情况下需要进入两条静脉时的抑制时间。
如果注射靠近尾部基部（更靠近小鼠身体）的侧静脉（特别是对于体重 >30 克的小鼠），请将注射角度从平行于静脉调整到与静脉 5°-10°，因为尾部底部的静脉比远端略深。
此处列出的 RNase 消化和 RNA 污染检查方案在从新鲜冷冻肝组织中分离的 DNA 样品上进行了验证，这些样品在 20 μL 中含有总共 175-700 ng 的核酸。还对从新鲜冷冻肝组织和 FACS 分选细胞中分离的 DNA 样品测试了 RNase 消化方案，以确认 RNase 消化后载体基因组和小鼠基因组的存在。使用目标扩增子的终点 PCR 扩增的琼脂糖凝胶电泳对结果进行可视化。
遵循所描述的方法可以减少掌握 IV 注射所需的培训和练习时间，并提高注射成功率，从而节省试剂。该协议使用简单且常用的工具，无需可能不容易获得的高级设备或设置。此外，此处列出的 IV 注射步骤可应用于需要静脉注射的各种注射液，例如反义寡核苷酸 （ASO），并根据注射液对注射液制备步骤进行适当修改。

单基因疾病占罕见病的 80%，共影响全球 3 亿人 1,2。目前还没有批准的治愈性疗法治疗大多数这些严重衰弱的罕见疾病 1,2,3。然而，单基因疾病是基因疗法的理想候选者，可以替代、补充、纠正或沉默功能失调的基因 4,5。目前，正在开发多种载体并将其用于向特定细胞类型递送基因疗法 4,6。其中一种载体是腺相关病毒 （AAV）。AAV 是一种非致病性细小病毒，越来越多地被用作基因治疗载体7。与其他病毒载体相比，AAV 具有较低的免疫原性、整合到宿主基因组中的可能性较低，并且能够有效转导各种组织中的分裂和非分裂细胞 7,8。此外，已经开发了多种方法来设计和鉴定具有理想特性的 AAV，例如特异性组织嗜性或进一步降低的免疫原性，这大大增强了 AAV 作为不同适应症的病毒载体的多功能性9。这些因素使 AAV 成为广泛研究的基因治疗载体，并导致多种 FDA 批准的基于 AAV 的基因疗法的开发10。
小鼠模型通常用于在体内测试潜在的基因疗法，并更好地了解单基因疾病的病理机制。这是由于小鼠模型对不同病症的病理学的概括，它们的基因组与人类基因组的相似性，以及小鼠处理、维护和生成相对容易 11,12,13。在研究影响身体多个系统或区域的疾病（例如肌营养不良）时，体内检测尤为重要。对于这些疾病，体外测试可能不足以全面评估全身给药后旨在到达不同身体区域的治疗的安全性、有效性、药代动力学和药效学14。
各种全身给药途径可用于给药。每种途径都有其优点、缺点以及与所研究的动物模型和药物的兼容性程度15。静脉内 （IV） 侧尾静脉注射是小鼠全身输送 AAV 的常用途径16。侧尾静脉注射允许快速、直接地将注射液注射到小鼠血液中，确保药物在体循环中的高生物利用度17。它们还需要执行相对简单且常用的工具。然而，主要是由于尾静脉直径小且难以定位静脉，外侧尾静脉注射在技术上具有挑战性，需要高度的技能和不断的练习，以避免注射尝试失败或剂量输送不完全 16,17,18,19。这些可能导致昂贵试剂的损失或结果不准确，尤其是在执行注射时未识别不完全注射的情况下。我们在这里总结的经验基于我们有据可查的文章中报道的协议，我们已针对我们的使用进行了调整，优化了外侧尾静脉注射程序的各个步骤，以确保始终如一地成功注射20、21、22、23、24、25、26、27。
在这里，我们描述了这种详细的优化侧尾静脉注射方案，以使用简单且常用的工具将 AAV 输送到未镇静的 7-9 周龄小鼠中。此外，我们还提供了用于评估 AAV 递送和生物分布的方法的方案。这些方案涵盖注射后组织收集、组织固定、DNA 提取和每个二倍体基因组 （vg/dg） 定量的数字聚合酶链反应 （dPCR） 载体基因组。此处提供的 IV 注射方案和指南旨在提高成功进行侧尾静脉注射的便利性。这可能有助于减少掌握注射技能所需的时间，同时提高注射的准确性和一致性。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.22 &#181;m syringe filter</td>
			<td>Millipore</td>
			<td>SLGVM33RS</td>
			<td></td>
		</tr>
		<tr>
			<td>0.3 mL insulin syringes with 29G needle</td>
			<td>BD Biosciences</td>
			<td>324702</td>
			<td></td>
		</tr>
		<tr>
			<td>1.7 mL microcentrifuge&#160; tube</td>
			<td>Crystalgen</td>
			<td>23-2051</td>
			<td></td>
		</tr>
		<tr>
			<td>10 mL syringe</td>
			<td>BD Biosciences</td>
			<td>302995</td>
			<td></td>
		</tr>
		<tr>
			<td>100% EtOH</td>
			<td>The Warner Graham Company</td>
			<td>201096</td>
			<td></td>
		</tr>
		<tr>
			<td>10x phosphate-buffered saline (PBS)</td>
			<td>Corning</td>
			<td>46-013-CM</td>
			<td>Used to prepare 1x PBS for tissue fixation</td>
		</tr>
		<tr>
			<td>15 mL conical tube</td>
			<td>Corning</td>
			<td>430766</td>
			<td></td>
		</tr>
		<tr>
			<td>15 mL conical tube holder</td>
			<td>Multiple sources</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>190 proof ethyl alcohol</td>
			<td>The Warner Graham Company</td>
			<td>6810-01-113-7320</td>
			<td>Used to prepare 70% ethanol</td>
		</tr>
		<tr>
			<td>1x sterile PBS</td>
			<td>Gibco</td>
			<td>10010023</td>
			<td></td>
		</tr>
		<tr>
			<td>2 mL microcentrifuge tissue storage tubes</td>
			<td>Eppendorf</td>
			<td>022363344</td>
			<td></td>
		</tr>
		<tr>
			<td>4% paraformaldehyde (PFA)</td>
			<td>Electron Microscopy Sciences</td>
			<td>157-4</td>
			<td></td>
		</tr>
		<tr>
			<td>Adeno-associated virus (AAV)</td>
			<td>Charles River</td>
			<td>N/A</td>
			<td>Single-stranded DNA (ssDNA) AAV was packaged to deliver Cre recombinase as the transgene driven by CMV promoter</td>
		</tr>
		<tr>
			<td>Alcohol swab</td>
			<td>BD Biosciences</td>
			<td>326895</td>
			<td></td>
		</tr>
		<tr>
			<td>Bead lysis tube</td>
			<td>Next Advance</td>
			<td>GREENE5</td>
			<td></td>
		</tr>
		<tr>
			<td>BsuRI (HaeIII) restriction enzyme</td>
			<td>Thermo Fisher Scientific</td>
			<td>ER0151&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Bullet blender</td>
			<td>Next Advance</td>
			<td>BBX24B</td>
			<td></td>
		</tr>
		<tr>
			<td>Ai14-derived mice from JAX 007914 strain (genetic background: C57BL/6J)</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>Mice containing Ai14 Cre-reporter allele were purchased from JAX (catalog number: 007914)</td>
		</tr>
		<tr>
			<td>Disposable absorbent pads</td>
			<td>Fisherbrand</td>
			<td>1420662</td>
			<td></td>
		</tr>
		<tr>
			<td>Dissection forceps</td>
			<td>Fine Science Tools (F.S.T)</td>
			<td>11251-35</td>
			<td></td>
		</tr>
		<tr>
			<td>Dissection scissors</td>
			<td>Fine Science Tools (F.S.T)</td>
			<td>14085-08</td>
			<td></td>
		</tr>
		<tr>
			<td>DNA degradation reagent (DNAZap)</td>
			<td>Invitrogen</td>
			<td>AM9890</td>
			<td></td>
		</tr>
		<tr>
			<td>DNA-Extraction RNase A</td>
			<td>Qiagen</td>
			<td>19101</td>
			<td>For RNA digestion during nucleic acid extraction</td>
		</tr>
		<tr>
			<td>DNase-free RNase for DNA cleanup</td>
			<td>&#160;F. Hoffmann-La Roche</td>
			<td>11119915001</td>
			<td>For RNA digestion after nucleic acid extraction</td>
		</tr>
		<tr>
			<td>dPCR Probe PCR Kit</td>
			<td>Qiagen</td>
			<td>250102</td>
			<td></td>
		</tr>
		<tr>
			<td>dPCR software&#160;</td>
			<td>Qiagen&#160;</td>
			<td>N/A&#160;</td>
			<td>QIAcuity Software Suite&#160;</td>
		</tr>
		<tr>
			<td>Elevated platform</td>
			<td>Multiple sources</td>
			<td>N/A</td>
			<td>An empty pipette tips box was used to elevate the mouse restrainer during tail warming up</td>
		</tr>
		<tr>
			<td>Fluorescence microscope</td>
			<td>Multiple sources</td>
			<td>N/A</td>
			<td>Model used here: Nikon Eclipse Ti</td>
		</tr>
		<tr>
			<td>Fluorescence microscope software&#160;</td>
			<td>Multiple sources&#160;</td>
			<td>N/A&#160;</td>
			<td>Software used here: NIS-Elements&#160;</td>
		</tr>
		<tr>
			<td>Gauze</td>
			<td>Covidien</td>
			<td>9022</td>
			<td></td>
		</tr>
		<tr>
			<td>Heat block</td>
			<td>Eppendorf</td>
			<td>Thermomixer 5350</td>
			<td></td>
		</tr>
		<tr>
			<td>High-speed centrifuge</td>
			<td>Eppendorf</td>
			<td>22620689</td>
			<td></td>
		</tr>
		<tr>
			<td>Metal container</td>
			<td>Vollrath</td>
			<td>80125</td>
			<td></td>
		</tr>
		<tr>
			<td>Methylbutane</td>
			<td>J.T. Baker</td>
			<td>Q223-08</td>
			<td></td>
		</tr>
		<tr>
			<td>Molecular grade water</td>
			<td>Quality Biological</td>
			<td>351-029-131</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse tube restrainer</td>
			<td>Braintree Scientific</td>
			<td>TV-RED-150-STD</td>
			<td></td>
		</tr>
		<tr>
			<td>Myfuge mini centrifuge</td>
			<td>Benchmark Scientific</td>
			<td>C1012</td>
			<td></td>
		</tr>
		<tr>
			<td>Polymerase chain reaction thermal cycler</td>
			<td>Bio-Rad Laboratories</td>
			<td>1851148</td>
			<td>Model: C1000 Touch</td>
		</tr>
		<tr>
			<td>Precision wipes</td>
			<td>Kimberly-Clark Professional</td>
			<td>7552</td>
			<td></td>
		</tr>
		<tr>
			<td>Proteinase K</td>
			<td>Qiagen</td>
			<td>19131</td>
			<td></td>
		</tr>
		<tr>
			<td>QIAcuity dPCR Nanoplate 26k 24-well</td>
			<td>Qiagen</td>
			<td>250001</td>
			<td></td>
		</tr>
		<tr>
			<td>QIAcuity One dPCR system</td>
			<td>Qiagen</td>
			<td>911020</td>
			<td></td>
		</tr>
		<tr>
			<td>Qiagen DNeasy Blood &#38; Tissue Kit</td>
			<td>Qiagen</td>
			<td>69504</td>
			<td>Used for DNA extraction from tissues</td>
		</tr>
		<tr>
			<td>Qiagen QIAamp DNA Micro Kit</td>
			<td>Qiagen</td>
			<td>56304</td>
			<td>Used for cleanup of genomic DNA, and the isolation of DNA from small volumes of blood prtocotol was used for DNA extraction from FACS-sorted cells</td>
		</tr>
		<tr>
			<td>Rodent restrainer cone</td>
			<td>Braintree Scientific</td>
			<td>MDC-200</td>
			<td></td>
		</tr>
		<tr>
			<td>Scale</td>
			<td>Ohaus</td>
			<td>72212663</td>
			<td></td>
		</tr>
		<tr>
			<td>Styrofoam box</td>
			<td>Multiple sources</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>Sigma-Aldrich</td>
			<td>S9378-1kg</td>
			<td></td>
		</tr>
		<tr>
			<td>Surface cleaner and disinfectant</td>
			<td>Peroxigard</td>
			<td>29101&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Timer</td>
			<td>Multiple sources</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Transfer forceps</td>
			<td>Fine Science Tools (F.S.T)</td>
			<td>91113-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Vortex</td>
			<td>Daigger &#38; Company</td>
			<td>22220A</td>
			<td>Model: Daigger Vortex Genie 2</td>
		</tr>
	</tbody>
</table>

consistent delivery of adeno-associated virus via lateral tail-vein injection in adult mice

许多疾病影响多个器官或涉及身体的不同区域，因此系统性地提供治疗以靶向位于不同部位的受影响细胞至关重要。静脉注射是临床前研究中广泛使用的全身给药途径，这些研究旨在评估全身给药的治疗方法。在成年小鼠中，它涉及将治疗剂静脉注射到小鼠的侧尾静脉中。掌握后，尾静脉注射既安全又快速，只需要简单且常用的工具。然而，尾静脉注射在技术上具有挑战性，需要广泛的培训和持续实践，以确保准确输送预期剂量。
在这里，我们描述了一个详细的、优化的、侧尾静脉注射方案，该方案是我们根据我们的经验和其他小组之前报告的建议制定的。除了鼠标限制器和胰岛素注射器外，该方案只需要大多数实验室中容易获得的试剂和设备。我们发现，遵循该方案可始终成功地将腺相关病毒 （AAV） 静脉内输送到未镇静的 7-9 周龄小鼠的尾静脉中。此外，我们描述了用于组织学检测荧光报告蛋白和每个二倍体基因组 （vg/dg） 定量载体基因组的优化方案，用于评估 AAV 转导和生物分布。该协议的目标是帮助实验者轻松成功且一致地进行尾静脉注射，这可以减少掌握该技术所需的练习时间。

Monogenic disorders make up 80% of rare diseases, which collectively affect 300 million individuals worldwide1,2. There are currently no approved curative therapies for the majority of these greatly debilitating rare disorders1,2,3. However, monogenic disorders are ideal candidates for gene therapies that can replace, supplement, correct, or silence dysfunctional genes4,5. Currently, multiple vectors are being developed and used to deliver gene therapies to specific cell types4,6. One of those vectors is adeno-associated virus (AAV). AAV is a non-pathogenic parvovirus that is increasingly being used as a gene therapy vector7. Compared to other viral vectors, AAV has lower immunogenicity, lower potential to integrate into the host genome, and the ability to efficiently transduce dividing and non-dividing cells in various tissues7,8. Additionally, multiple approaches have been developed to engineer and identify AAVs with desirable characteristics such as specific tissue tropism or further reduced immunogenicity, which greatly enhances AAV&#39;s versatility as a viral vector for different indications9. These factors have made AAV a widely investigated gene therapy vector and led to the development of multiple FDA-approved AAV-based gene therapies10.
Mouse models are commonly used to test potential gene therapies in vivo and better understand the pathomechanisms of monogenic disorders. This is due to the mouse models&#39; recapitulation of the pathologies of different conditions, their genome&#39;s similarity to the human genome, and the relative ease of mouse handling, maintenance, and generation11,12,13. In vivo testing is particularly important when studying disorders that affect multiple systems or regions of the body, such as muscular dystrophies. For these disorders, in vitro testing might not be sufficient to comprehensively assess the safety, efficacy, pharmacokinetics, and pharmacodynamics of therapeutics intended to reach different body regions after systemic administration14.
Various systemic administration routes can be used to deliver drugs. Each route has its advantages, drawbacks, and degree of compatibility with the animal model and drug being investigated15. Intravenous (IV) lateral tail-vein injection is a commonly used route for systemic delivery of AAV in mice16. Lateral tail-vein injections allow fast and direct administration of the injectate into the mouse bloodstream ensuring high drug bioavailability in systemic circulation17. They also require relatively simple and commonly available tools to be performed. However, mainly due to the small tail vein diameter and difficulty in locating the vein, lateral tail-vein injections are technically challenging and require a high degree of skill and constant practice to avoid failed injection attempts or incomplete dose delivery16,17,18,19. These can result in the loss of expensive reagents or inaccurate results, especially if the incomplete injection is not recognized while performing the injection. Our experience summarized here is based on protocols reported in well-documented articles that we have adapted for our use, optimizing various steps of the lateral tail-vein injection procedure to ensure consistently successful injections20,21,22,23,24,25,26,27.
Here, we describe this detailed optimized lateral tail-vein injection protocol to deliver AAV into unsedated 7-9-week-old mice using simple and commonly available tools. Additionally, we provide the protocols for methods used to assess AAV delivery and biodistribution. These protocols cover post injection tissue collection, tissue fixation, DNA extraction, and digital polymerase chain reaction (dPCR) vector genome per diploid genome (vg/dg) quantification. The IV injection protocol and pointers provided here aim to enhance the ease of successfully performing lateral tail-vein injections. This will potentially help reduce the time needed to master the injection skills while simultaneously improving the accuracy and consistency of injections.

作者聚焦：开发针对肌营养不良症和神经肌肉疾病的基因疗法

通过侧尾静脉注射在成年小鼠中持续递送腺相关病毒

7 至 9 周龄雄性小鼠通过侧尾静脉注射以 1.5 ×10 12 vg/小鼠注射 AAV，注射液量为 150-200 μL。这里使用的 ssDNA AAV 递送了由 CMV 启动子驱动的 Cre 重组酶转基因。注射的小鼠是 Cre 报告基因 Ai14 等位基因的纯合子。当暴露于 Cre 重组酶时，含有 Ai14 等位基因的细胞表达荧光 tdTomato 蛋白。由于 tdTomato 表达是由 Cre 诱导的基因组重组引起的，因此表达 tdTomato 的细胞表示由 AAV 直接转导的细胞或转导细胞的后代细胞。此处显示的数据是以 1.5 × 1012 vg/小鼠注射 AAV9-CMV-Cre 的小鼠，以 160 μL （5.8-5.9 × 1013 vg/kg） 给药。注射后 28 天处死小鼠，如上所述收集组织。消化少量骨骼肌和肝叶，并使用 FACS 收集其细胞。立即使用预冷的甲基丁烷冷冻几个肝叶进行核酸提取。将一些骨骼肌和肝叶固定冷冻用于荧光 tdTomato 的组织学成像。tdTomato 在整个肝脏（图 2A）和股四头肌（图 2C）中弥漫表达，表明 AAV9 广泛到达并转导了两个组织的不同区域。
从新鲜冷冻肝脏和 FACS 分选细胞中提取的 DNA 用于通过 dPCR 定量 vg/dg。Vg/dg 定量可用于评估分析样品中 AAV 的进样一致性和转导效率。使用来自新鲜冷冻肝组织样品和 FACS 分选细胞的 1D 液滴散点图来确保测定的有效性（图 3A、C）。散点图显示存在阳性和阴性分区，阳性和阴性分区之间的明确分离，可以准确确定检测阈值，以及阳性和阴性分区之间存在少量液滴，这可能会降低 dPCR 检测的准确性。满足所有这些标准表明 dPCR 检测结果是有效的。定量每个样品中 Polr2a 基因拷贝数以确定小鼠二倍体基因组（2 个 Polr2a 基因拷贝/小鼠二倍体基因组）的数量，并使用针对 Cre 重组酶转基因序列的引物/探针来定量病毒基因组（1 个转基因拷贝/病毒基因组， 表 1）。对新鲜冷冻肝组织样品和 FACS 分选细胞的 vg/dg 值进行量化，并显示每个样品中分别存在 187.7 vg/dg 和 4.7 vg/dg（图 3B、D）。来自 PBS 注射小鼠的样品和不含核酸的非模板对照用作阴性对照。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/66934/66934fig01.jpg"> 图 1：静脉注射站概述。 （A） 进行 IV 注射所需的工具。此处显示的是 （1） 计时器、（2） 小鼠管限制器、（3） 未切割和 （4） 切割的塑料限制锥、（5） 酒精棉签、（6） 用作升高小鼠管限制器平台的空移液器吸头盒、（7） 一次性吸收垫、（8） 15 mL 温水锥形管、（9） 15 mL 管架、（10） 纱布和 （11） 胰岛素注射器。（B） 首先将鼠标放入管式限制器内。然后，如果鼠标太小而无法仅由管式限制器约束，则插入切割的限制锥体以在鼠标周围形成一个限制套。确保鼠标的呼吸不受限制器的阻碍。管式限制器放置在高架平台的顶部，以便将小鼠尾巴放入温水中。（C） 注射前的小鼠尾巴位置和针头保持角度。向后拉尾部，使尾部伸展，注射部位完全水平。针平行于尾部和静脉，斜面朝上。 <a href="https://www.jove.com/files/ftp_upload/66934/66934fig01large.jpg" target="_blank">请单击此处查看此图的较大版本。</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/66934/66934fig02.jpg"> 图 2：IV 注射后荧光报告蛋白的检测。 携带 Cre 报告基因 Ai14 等位基因的 7 至 9 周龄雄性小鼠静脉注射 1.5 ×10 12 vg/小鼠的 AAV9-CMV-Cre，以 160 μL（5.8-5.9 ×10 13 vg/kg）或 PBS 给药。AAV9 输送 Cre IV 注射后小鼠 （A） 肝脏或 （C） 股四头肌切片的代表性荧光图像。（B） 对注射 PBS 的小鼠的肝脏或 （D） 股四头肌切片进行成像，作为阴性对照。收集组织并在 IV 注射后 28 天固定冷冻。Cre 暴露后，荧光 tdTomato 蛋白在转导细胞和转导细胞的后代细胞中表达。以 10 倍放大倍率对 10 μm 厚的切片进行成像。比例尺 = 100 μm。 <a href="https://www.jove.com/files/ftp_upload/66934/66934fig02large.jpg" target="_blank">请点击此处查看此图的较大版本。</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/66934/66934fig03.jpg"> 图 3：每个二倍体基因组 （vg/dg） 定量的载体基因组。 从注射 AAV9-CMV-Cre 或 PBS 的小鼠中收集的 （A） 肝组织或 （C） FACS 分选细胞中 dPCR 载体基因组定量的 1D 散点图。散点图显示了阳性和阴性 dPCR 分配，以及样品上水平线指示的检测阈值。（B，D） 定量 （B） 肝组织或 （D） FACS 分选细胞样品中的小鼠二倍体基因组和载体基因组后，进行 vg/dg 定量。此处显示的结果来自一只注射 AAV9 的小鼠和一只注射 PBS 的小鼠，每只小鼠都有一个技术 dPCR 副本。误差线表示每个样本的 95% 置信区间。缩写： NTC= 非模板对照;dPCR = 数字 PCR;FACS = 荧光激活细胞分选。 <a href="https://www.jove.com/files/ftp_upload/66934/66934fig03large.jpg" target="_blank">请单击此处查看此图的较大版本。</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td>底漆</td>
			<td>序列</td>
		</tr><tr><td>Cre 正向引物</td>
			<td>CTGACGGTGGGAGAATGTTAAT</td>
		</tr><tr><td>Cre 反向引物</td>
			<td>CATCGCTCGACCAGTTTAGTT</td>
		</tr><tr><td>Cre 探针</td>
			<td>/56-FAM/CGCAGGTGT/ZEN/AGAGAAGGCACTTAGC/3IABkFQ/</td>
		</tr><tr><td>Polr2a 正向引物</td>
			<td>GACTCCTTCACTCACTGTCTTC</td>
		</tr><tr><td>Polr2a 反向引物</td>
			<td>TCTTGCTAGGCAGTCCATTATC</td>
		</tr><tr><td>Polr2a 探针</td>
			<td>/5HEX/ACGAGATGC/ZEN/TGAAAGAGCCAAGGT/3IABkFQ/</td>
		</tr></tbody></table>表 1：用于 vg/dg 定量的引物和探针序列。 Cre 引物和探针用于定量载体基因组。Polr2a 引物和探针用于定量小鼠二倍体基因组。

在这里，我们详细介绍了小鼠侧尾静脉注射的优化方案，以在成年小鼠中全身施用腺相关病毒 （AAV）。此外，我们描述了评估 AAV 转导的常用检测方案。

Many disorders affect multiple organs or involve different regions of the body, so it is critical to deliver therapeutics systemically to target the ...

consistent-delivery-adeno-associated-virus-via-lateral-tail-vein

Research

JoVE Journal

Medicine

Consistent Delivery of Adeno-Associated Virus via Lateral Tail-Vein Injection in Adult Mice

604 次观看.  National Institutes of Health. 我们致力于开发针对先天性和早发性肌营养不良症和神经肌肉疾病的基因疗法。为了实现这一目标，我们使用不同的基因治疗方式来解决这些疾病的潜在遗传原因。病毒和非病毒基因治疗载体的不断发展对我们的领域非常重要。由于新的改进病毒和非病毒基因治疗载体允许开发更安全、更高效的基因疗法，这反过来又提高了安全性并缩短了为患...

视频: 作者聚焦：开发针对肌营养不良症和神经肌肉疾病的基因疗法

Many disorders affect multiple organs or involve different regions of the body, so it is critical to deliver therapeutics systemically to target the affected cells located in different sites. Intravenous injection is a widely used systemic delivery route in preclinical studies that assess treatments intended for body-wide administration. In adult mice, it involves the intravenous administration of the therapeutic agent into the mouse&#39;s lateral tail veins. When mastered, tail-vein injections are safe and fast, and only require simple and commonly available tools. However, tail-vein injections are technically challenging and require extensive training and continuous practice to ensure the accurate delivery of the intended dose.
Here we describe a detailed, optimized, lateral tail-vein injection protocol that we have developed based on our experience and on recommendations that had been previously reported by other groups. Other than the mouse restrainers and insulin syringes, this protocol requires only reagents and equipment that are readily available in most labs. We found that following this protocol results in consistently successful intravenous delivery of adeno-associated virus (AAV) into the tail veins of unsedated 7-9 week-old mice. Additionally, we describe the optimized protocols for the histological detection of fluorescent reporter proteins and vector genome per diploid genome (vg/dg) quantification used to assess AAV transduction and biodistribution. The goal of this protocol is to aid experimenters in easily performing tail-vein injections successfully and consistently, which can reduce the practice time needed to master the technique.

Here we detail an optimized protocol for mouse lateral tail-vein injection to systemically administer adeno-associated virus (AAV) in adult mice. Additionally, we describe protocols of commonly used assays to assess AAV transduction.