Name: digital droplet pcr method for the quantification of aav transduction efficiency in murine retina
Uploaded: 2021-12-25T14:00:00
Description: Watch this Scientific Journal Video about Digital Droplet PCR Method for the Quantification of AAV Transduction Efficiency in Murine Retina at JoVE.com

我们要感谢Oezkan Keles，Josephine Jüttner和巴塞尔分子与临床眼科研究所的Botond Roska教授，复杂病毒平台对AAV生产的帮助和支持。我们还要感谢宾夕法尼亚大学佩雷尔曼医学院的Jean Bennett教授的AAV8 / BP2菌株。动物工作在盖布泽技术大学动物设施进行。为此，我们感谢Leyla Dikmetas和Uygar Halis Tazebay教授对畜牧业的技术援助和支持。我们还要感谢Fatma Ozdemir博士对手稿的评论。这项工作得到了TUBITAK，授权号118C226和121N275以及Sabanci University Integration grant的支持。

所有实验方案均被萨班哲大学伦理委员会接受，实验按照"视觉和眼科研究协会"的声明进行，用于研究动物
1. 小规模AAV生产12
<ol><li>使用15cm板在完整的10 mL DMEM / 10%FBS中培养HEK293T细胞，直到70-80%汇合。</li>
	<li>用20μg辅助质粒（pHGT1-Adeno1），7μg衣壳质粒和7μgAAV2-CBA-tdTomato-WPRE载体和136μLPEI溶液（1mg / mL）在5mL DMEM中制备转染混合物。</li>
	<li>将5mL?...

<ol>
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H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Controlling+AAV+tropism+in+the+nervous+system+with+natural+and+engineered+capsids.">Controlling AAV tropism in the nervous system with natural and engineered capsids.</a> Methods in Molecular Biology. 1382, 133-149 (2016).</li><li>Agbandje-McKenna, M., Kleinschmidt, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=AAV+capsid+structure+and+cell+interactions.">AAV capsid structure and cell interactions.</a> Methods in Molecular Biology. 807, 47-92 (2011).</li><li>Han, I. C., 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=Retinal+tropism+and+transduction+of+adeno-associated+virus+varies+by+serotype+and+route+of+delivery+(intravitreal,+subretinal,+or+suprachoroidal)+in+rats.">Retinal tropism and transduction of adeno-associated virus varies by serotype and route of delivery (intravitreal, subretinal, or suprachoroidal) in rats.</a> Human Gene Therapy. 31 (23-24), 1288-1299 (2020).</li><li>Muraine, L., 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=Transduction+efficiency+of+adeno-associated+virus+serotypes+after+local+injection+in+mouse+and+human+skeletal+muscle.">Transduction efficiency of adeno-associated virus serotypes after local injection in mouse and human skeletal muscle.</a> Human Gene Therapy. 31 (3-4), 233-240 (2020).</li><li>Cronin, T., 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=Efficient+transduction+and+optogenetic+stimulation+of+retinal+bipolar+cells+by+a+synthetic+adeno-associated+virus+capsid+and+promoter.">Efficient transduction and optogenetic stimulation of retinal bipolar cells by a synthetic adeno-associated virus capsid and promoter.</a> EMBO Molecular Medicine. 6 (9), 1175-1190 (2014).</li><li>Higashimoto, T., 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+woodchuck+hepatitis+virus+post-transcriptional+regulatory+element+reduces+readthrough+transcription+from+retroviral+vectors.">The woodchuck hepatitis virus post-transcriptional regulatory element reduces readthrough transcription from retroviral vectors.</a> Gene Therapy. 14 (17), 1298-1304 (2007).</li><li>Pinheiro, L. B., 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=Evaluation+of+a+droplet+digital+polymerase+chain+reaction+format+for+DNA+copy+number+quantification.">Evaluation of a droplet digital polymerase chain reaction format for DNA copy number quantification.</a> Annals in Chemistry. 84 (2), 1003-1011 (2012).</li><li>Albayrak, C., 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=Digital+quantification+of+proteins+and+mrna+in+single+mammalian+cells.">Digital quantification of proteins and mrna in single mammalian cells.</a> Molecular Cell. 61 (6), 914-924 (2016).</li><li>Lock, M., Alvira, M. R., Chen, S. J., Wilson, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Absolute+determination+of+single-stranded+and+self-complementary+adeno-associated+viral+vector+genome+titers+by+droplet+digital+PCR.">Absolute determination of single-stranded and self-complementary adeno-associated viral vector genome titers by droplet digital PCR.</a> Hum Gene Therapy Methods. 25 (2), 115-125 (2014).</li><li>Juttner, J., 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=Targeting+neuronal+and+glial+cell+types+with+synthetic+promoter+AAVs+in+mice,+non-human+primates+and+humans.">Targeting neuronal and glial cell types with synthetic promoter AAVs in mice, non-human primates and humans.</a> Nature Neuroscience. 22 (8), 1345-1356 (2019).</li><li>Vandenberghe, L. H., 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=Efficient+serotype-dependent+release+of+functional+vector+into+the+culture+medium+during+adeno-associated+virus+manufacturing.">Efficient serotype-dependent release of functional vector into the culture medium during adeno-associated virus manufacturing.</a> Human Gene Therapy. 21 (10), 1251-1257 (2010).</li><li>Barben, M., Schori, C., Samardzija, M., Grimm, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeting+Hif1a+rescues+cone+degeneration+and+prevents+subretinal+neovascularization+in+a+model+of+chronic+hypoxia.">Targeting Hif1a rescues cone degeneration and prevents subretinal neovascularization in a model of chronic hypoxia.</a> Molecular Neurodegeneration. 13 (1), 12 (2018).</li><li>Chan, K. Y., 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=Engineered+AAVs+for+efficient+noninvasive+gene+delivery+to+the+central+and+peripheral+nervous+systems.">Engineered AAVs for efficient noninvasive gene delivery to the central and peripheral nervous systems.</a> Nature Neuroscience. 20 (8), 1172-1179 (2017).</li><li>Guiet, R., Burri, O., Seitz, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Open+source+tools+for+biological+image+analysis.">Open source tools for biological image analysis.</a> Methods Molecular Biology. 2040, 23-37 (2019).</li><li>Parks, M. M., 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=Variant+ribosomal+RNA+alleles+are+conserved+and+exhibit+tissue-specific+expression.">Variant ribosomal RNA alleles are conserved and exhibit tissue-specific expression.</a> Science Advances. 4 (2), (2018).</li><li>Aurnhammer, C., 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=Universal+real-time+PCR+for+the+detection+and+quantification+of+adeno-associated+virus+serotype+2-derived+inverted+terminal+repeat+sequences.">Universal real-time PCR for the detection and quantification of adeno-associated virus serotype 2-derived inverted terminal repeat sequences.</a> Human Gene Therapy Methods. 23 (1), 18-28 (2012).</li><li>Maclachlan, T. K., 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=Preclinical+safety+evaluation+of+AAV2-sFLT01-+a+gene+therapy+for+age-related+macular+degeneration.">Preclinical safety evaluation of AAV2-sFLT01- a gene therapy for age-related macular degeneration.</a> Molecular Therapy. 19 (2), 326-334 (2011).</li><li>Stieger, K., 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=Detection+of+intact+rAAV+particles+up+to+6+years+after+successful+gene+transfer+in+the+retina+of+dogs+and+primates.">Detection of intact rAAV particles up to 6 years after successful gene transfer in the retina of dogs and primates.</a> Molecular Therapy. 17 (3), 516-523 (2009).</li><li>Gyorgy, B., 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=Allele-specific+gene+editing+prevents+deafness+in+a+model+of+dominant+progressive+hearing+loss.">Allele-specific gene editing prevents deafness in a model of dominant progressive hearing loss.</a> Nature Medicine. 25 (7), 1123-1130 (2019).</li><li>Cox, D. B. T., 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=RNA+editing+with+CRISPR-Cas13.">RNA editing with CRISPR-Cas13.</a> Science. 358 (6366), 1019-1027 (2017).</li></ol>

在该协议中，我们生成了两个具有不同衣壳蛋白的AAV载体，然后相应地滴定它们。该协议最关键的步骤之一是产生足够量的AAV，这将在转导12，13之后产生可检测的报告者表达。
AAV的滴度也是调整玻璃体内注射AAV剂量的重要因素。一旦达到这些重要标准，就可以通过dd-PCR方法量化AAV的转导效率。
许多实验室正在...

腺相关病毒（AAV）现在通常用于各种视网膜基因治疗研究。AAV提供了一种安全有效的基因递送方式，免疫原性较低，基因组整合较少。AAV通过内吞作用进入靶细胞，这需要细胞表面受体和共受体结合1，2。因此，AAV对不同细胞类型的转导效率主要取决于衣壳及其与宿主细胞受体的相互作用。AAV具有血清型，每种血清型可以具有不同的细胞/组织向性和转导效率。还有人工AAV血清型和变异，通过化学修饰病毒衣壳，杂交衣壳的产生，肽插入，衣壳洗牌，定向进化和合理诱变产生3。即使氨基酸序列或衣壳结构的微小变化也会对与宿主细胞因子的相互作用产生影响，并导致不同的趋向性4。除了衣壳变异体外，其他因素，如注射途径和AAV生产的批次间变异，都会影响AAV在神经元视网膜中的转导效率。因此，需要可靠的方法来比较不同变体的转导速率。
大多数测定AAV转导效率的方法依赖于报告基因表达。这些包括荧光成像，免疫组织化学，蛋白质印迹或报告基因产物5，6，7的组织化学分析。然而，由于AAV的大小限制，包括报告基因来监测转导效率并不总是可行的。使用强启动子，如杂交CMV增强子/鸡β-肌动蛋白或Woodchuck肝炎转录后调节元件（WPRE）作为mRNA稳定剂序列，进一步使大小问题8复杂化。因此，使用更直接的方法定义注入的AAV的转导率也是有益的。
数字液滴PCR（dd-PCR）是一种从微量样品中定量靶DNA的强大技术。dd-PCR技术依赖于通过油滴封装靶DNA和PCR反应混合物。每个dd-PCR反应都含有数千个液滴。每个液滴作为独立的PCR反应进行处理和分析9。液滴分析只需使用泊松算法即可计算出任何样品中靶DNA分子的绝对拷贝数。由于AAV的转导效率与神经元视网膜中AAV基因组的拷贝数相关，因此我们使用dd-PCR方法量化AAV基因组。
在这里，我们描述了一种dd-PCR方法，用于计算来自视网膜基因组DNA6，10的AAV载体的转导效率。首先，使用小规模方案生成表达tdTomato报告的AAV，并通过dd-PCR方法11滴定。其次，AAV被玻璃体内注射到神经元视网膜中。为了证明转导效率，我们首先使用荧光显微镜和ImageJ软件量化了tdTomato表达。随后使用dd-PCR分离基因组DNA，用于定量注射视网膜中的AAV基因组。tdTomato表达水平与dd-PCR定量转导的AAV基因组的比较表明，dd-PCR方法准确量化了AAV载体的转导效率。我们的实验方案展示了一种基于dd-PCR的量化AAV转导效率的方法的详细说明。在该协议中，我们还显示了玻璃体内注射后转导的AAV基因组的绝对数量，只需使用基因组DNA分离和dd-PCR结果后的稀释因子即可。总体而言，该协议提供了一种强大的方法，该方法将成为报告表达的替代方法，以量化视网膜中AAV载体的转导效率。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>96-Well Semi-Skirted ddPCR plates</td>
			<td>BioRad</td>
			<td>12001925</td>
			<td>ddPCR</td>
		</tr>
		<tr>
			<td>Amicon Filter</td>
			<td>Millipore</td>
			<td>UFC910096</td>
			<td>AAV</td>
		</tr>
		<tr>
			<td>C1000 TOUCH 96 DEEP WELLS</td>
			<td>BioRad</td>
			<td>1851197</td>
			<td>ddPCR</td>
		</tr>
		<tr>
			<td>C57BL/6JRj mice strain</td>
			<td>Janvier</td>
			<td>C57BL/6JRj</td>
			<td>Mice</td>
		</tr>
		<tr>
			<td>DG8 gaskets</td>
			<td>BioRad</td>
			<td>1863009</td>
			<td>ddPCR</td>
		</tr>
		<tr>
			<td>DG8 Cartridges</td>
			<td>BioRad</td>
			<td>1864008</td>
			<td>ddPCR</td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Lonza</td>
			<td>BE12-604Q</td>
			<td>AAV</td>
		</tr>
		<tr>
			<td>DPBS</td>
			<td>PAN BIOTECH</td>
			<td>L 1825</td>
			<td>AAV</td>
		</tr>
		<tr>
			<td>Droplet generation oil eva green</td>
			<td>BioRad</td>
			<td>1864006</td>
			<td>ddPCR</td>
		</tr>
		<tr>
			<td>Droplet reader oil</td>
			<td>BioRad</td>
			<td>1863004</td>
			<td>ddPCR</td>
		</tr>
		<tr>
			<td>FBS</td>
			<td>PAN BIOTECH</td>
			<td>p30-3306</td>
			<td>AAV</td>
		</tr>
		<tr>
			<td>Foil seals for PX1 PCR Plate sealer</td>
			<td>BioRad</td>
			<td>1814040</td>
			<td>ddPCR</td>
		</tr>
		<tr>
			<td>Insulin Syringes</td>
			<td>BD Medical</td>
			<td>320933</td>
			<td>Intravitreal injection</td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>ADEKA <img alt="Equation 2" src="/files/ftp_upload/63038/63038eq02v2.jpg" />LA&#199; SANAY<img alt="Equation 2" src="/files/ftp_upload/63038/63038eq02v2.jpg" /> VE T<img alt="Equation 2" src="/files/ftp_upload/63038/63038eq02v2.jpg" />CARET A.<img alt="Equation 1" src="/files/ftp_upload/63038/63038eq01v2.jpg" />.</td>
			<td>N01AB06</td>
			<td>anesthetic</td>
		</tr>
		<tr>
			<td>Microinjector MM33</td>
			<td>World Precision Instruments</td>
			<td>82-42-101-0000</td>
			<td>Intravitreal injection</td>
		</tr>
		<tr>
			<td>Micron IV</td>
			<td>Phoenix Research Labs</td>
			<td>Micron IV</td>
			<td>Microscopy system based on 3-CCD color camera, frame grabber, and off-the-shelf software enables researchers to image mouse retinas.</td>
		</tr>
		<tr>
			<td>Mydfrin (%2.5 phenylephrine hydrochloride)</td>
			<td>Alcon</td>
			<td>S01FB01</td>
			<td>pupil dilation</td>
		</tr>
		<tr>
			<td>Nanofil Syringe 10 &#956;l</td>
			<td>World Precision Instruments</td>
			<td>NANOFIL</td>
			<td>Intravitreal injection</td>
		</tr>
		<tr>
			<td>Needle RN G36, 25 mm, PST 2</td>
			<td>World Precision Instruments</td>
			<td>NF36BL-2</td>
			<td>Intravitreal injection</td>
		</tr>
		<tr>
			<td>PEI-MAX</td>
			<td>Polyscience</td>
			<td>24765-1</td>
			<td>AAV</td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin</td>
			<td>PAN BIOTECH</td>
			<td>P06-07100</td>
			<td>AAV</td>
		</tr>
		<tr>
			<td>Plasmid pHGT1-Adeno1</td>
			<td>PlasmidFactory</td>
			<td>PF1236</td>
			<td>AAV</td>
		</tr>
		<tr>
			<td>Pluronic F-68</td>
			<td>Gibco</td>
			<td>24040032</td>
			<td>AAV</td>
		</tr>
		<tr>
			<td>PX1 PCR Plate Sealer system</td>
			<td>BioRad</td>
			<td>1814000</td>
			<td>ddPCR</td>
		</tr>
		<tr>
			<td>QX200 ddPCR EvaGreen Supermix</td>
			<td>BioRad</td>
			<td>1864034</td>
			<td>ddPCR</td>
		</tr>
		<tr>
			<td>QX200 Droplet Reader/QX200 Droplet Generator</td>
			<td>BioRad</td>
			<td>1864001</td>
			<td>ddPCR</td>
		</tr>
		<tr>
			<td>SPLITTER FORCEP WATCHER 
			MAKER - LENGTH = 13.5 CM</td>
			<td>endostall medical</td>
			<td>EJN-160-0155</td>
			<td>Retina isolation</td>
		</tr>
		<tr>
			<td>Steril Syringe Filter</td>
			<td>AISIMO</td>
			<td>ASF33PS22S</td>
			<td>AAV</td>
		</tr>
		<tr>
			<td>Tissue Genomic DNA Kit</td>
			<td>EcoSpin</td>
			<td>E1070</td>
			<td>gDNA isolation</td>
		</tr>
		<tr>
			<td>Tobradex (0.3% tobramycin / 0.1% dexamethasone)</td>
			<td>Alcon</td>
			<td>S01CA01</td>
			<td>anti-inflammatory / antibiotic</td>
		</tr>
		<tr>
			<td>Tropamid (% 0.5 tropicamide)</td>
			<td>Bilim <img alt="Equation 2" src="/files/ftp_upload/63038/63038eq02v2.jpg" />la&#231; Sanayi ve Ticaret A.<img alt="Equation 1" src="/files/ftp_upload/63038/63038eq01v2.jpg" />.</td>
			<td>S01FA06</td>
			<td>pupil dilation</td>
		</tr>
		<tr>
			<td>Turbonuclease</td>
			<td>Accelagen</td>
			<td>N0103L</td>
			<td>AAV</td>
		</tr>
		<tr>
			<td>Viscotears (carbomer 2 mg/g)</td>
			<td>Bausch+Lomb</td>
			<td>S01XA20</td>
			<td>lubricant eye drop</td>
		</tr>
	</tbody>
</table>

digital droplet pcr method for the quantification of aav transduction efficiency in murine retina

许多视网膜细胞生物学实验室现在经常使用腺相关病毒（AAV）进行基因编辑和调控应用。AAV转导的效率通常是至关重要的，这会影响整体实验结果。转导效率的主要决定因素之一是AAV载体的血清型或变体。 目前，各种人工AAV血清型和变体可用于宿主细胞表面受体的不同亲和力。对于视网膜基因治疗，这导致不同视网膜细胞类型的转导效率不同。此外，注射路线和AAV生产的质量也可能影响视网膜AAV转导效率。因此，比较不同变体、批次和方法的效率至关重要。数字液滴PCR（dd-PCR）方法以高精度定量核酸，并允许在没有任何标准品或参考的情况下对给定靶标进行绝对定量。使用dd-PCR，通过绝对定量注射视网膜内的AAV基因组拷贝数来评估AAV的转导效率也是可行的。在这里，我们提供了一种使用dd-PCR量化视网膜细胞中AAV转导率的简单方法。通过微小的修改，这种方法也可以成为线粒体DNA拷贝数定量的基础，以及评估碱基编辑的效率，这对于几种视网膜疾病和基因治疗应用至关重要。

小规模AAV生产是一种快速有效的方法，可为玻璃体内注射提供载体（图1）。小规模AAV生产通常使滴度在1 x 1012 GC / ml的范围内，足以检测视网膜中的报告表达（图2）。使用滴定聚合酶链反应对 AAV 进行滴定可提供一致的结果。常规使用ITR2和WPRE特异性引物，并使用dd-PCR软件通过建模为泊松分布来计算每个靶分子的起始浓度。通过增殖稀释因子并?...

Watch this Scientific Journal Video about Digital Droplet PCR Method for the Quantification of AAV Transduction Efficiency in Murine Retina at JoVE.com

Digital Droplet PCR Method for the Quantification of AAV Transduction Efficiency in Murine Retina

该协议介绍了如何使用数字液滴PCR（dd-PCR）以及小规模AAV生产，玻璃体内注射，视网膜成像和视网膜基因组DNA分离来量化小鼠视网膜中的AAV转导效率。

数字液滴PCR法定量分析小鼠视网膜AAV转导效率

Many retinal cell biology laboratories now routinely use Adeno-associated viruses (AAVs) for gene editing and regulatory applications. The efficiency of ...

在本视频中，我们将展示如何使用数字液滴PCR方法量化小鼠视网膜中的AAV转导，该方法非常灵敏，可以对目标DNA进行绝对定量。我们开发了这种方法，主要用于大型AVV构建体，不允许我们使用像基于CRISPR的构建体这样的报告基因。通过使用这种方法，我们可以简单地量化视网膜中AAV的转导率。该方法依赖于数字液滴PCR的灵敏度和绝对定量，并产生直接的结果。在本视频中，我们将展示如何使用数字液滴PCR方法量化小鼠视网膜中的AAV转导，该方法非常灵敏，可以对目标DNA进行绝对定量。我们已经开发了这种方法，主要用于大面积构建体，不允许我们使用像基于CRISPR的构建体这样的报告基因。通过使用这种方法，我们可以简单地量化视网膜中AAV的转导率。该方法依赖于两个液滴PCR的灵敏度和绝对定量，并产生直接的结果。由于数字液滴PCR方法的效率，一些实验室已经从定量实时荧光定量PCR转向数字液滴PCR或AAV滴定。该方法还可以为视网膜中的线粒体DNA定量提供基础，以及检查基于CRISPR的突变校正对小鼠视网膜基因治疗的效率。用AAV辅助质粒，AAV衣壳质粒，AAV载体和PI溶液制备5毫升所需的转染混合物。将制备的5毫升转染混合物加入培养基中。在37摄氏度下孵育转染的细胞。在转染后 48 至 60 小时收集培养基。用DNA以每毫升250单位的终浓度在37摄氏度下消化30分钟，并在4， 000 g 4摄氏度下离心30分钟。用PBS预润湿再生纤维素膜。用0.22微米注射器过滤器过滤介质，放入预润湿的再生纤维素膜中，共过滤100千道尔顿。将再生纤维素膜在4，000 G四摄氏度下离心30分钟并丢弃培养基。用含有0.001%pluronic F68的PBS洗涤再生纤维素膜并在4000 G 4摄氏度下离心三次30分钟。从再生纤维素膜的顶部收集浓缩的AAV并分配以供进一步使用。在这里，我们展示了如何进行小规模的AAV生产。在审查此协议后，您应该能够制作足以看到视网膜中报告者表达的AVV。您也可以遵循数字PCR的实验方案，以便能够滴定产生的AAV。麻醉前涂抹一滴含有0.5%托吡卡胺和2.5%去氧肾上腺素盐酸盐的眼药水，以扩张瞳孔。用异氟醚麻醉小鼠并验证反射。在注射程序之前，将0.3%妥布霉素和0.1%地塞米松无菌眼药溶液滴眼液涂抹在每只眼睛上。使用手术钩稳定上眼睑。稍微向后拉，露出眼睛的背侧部分。将挂钩用胶带固定在带上以保持到位。用弯曲的虹膜剪刀去除结膜，露出眼睛的巩膜。使用新鲜无菌的30G胰岛素注射器刺穿巩膜。使用微型机械手将微型注射器的针头通过相同的穿刺插入。将针尖放在眼罩中间的晶状体后面。将一微升AAV溶液缓慢注入眼睛的玻璃体中。将针头留在原位一分钟，然后慢慢取出针头。将含有0.3%妥布霉素和0.1%地塞米松的抗炎和抗菌局部凝胶治疗应用于眼罩。涂抹一滴0.5%托吡卡胺。和含有2.5%去氧肾上腺素盐酸盐的滴眼液在麻醉前扩张瞳孔。将每克外用凝胶2毫克的尿素涂抹在眼睛表面。根据需要调整鼠标支架以将鼠标眼睛居中。在玻璃体内注射后一周和两周对双眼进行眼底和荧光成像，以跟踪TdTomato表达。在手术前使用二氧化碳吸入对动物实施安乐死。在13.5厘米的分离器镊子的帮助下将眼球向前移动。取出晶状体和剩余的玻璃体。用精密弯曲的镊子切断眼球与视神经的连接，并使用相同的弯曲镊子轻轻挤压视网膜。使用商业化的蛋白酶K消化膜组织基因组DNA试剂盒分离基因组DNA。用蛋白酶K.Lyse视网膜在55摄氏度200 G下完全消化视网膜30分钟，并遵循制造商的protocol。在这些实验方案中，我们显示了玻璃体内注射眼底荧光成像，以及视网膜和基因组DNA分离。这些是视网膜非常关键的程序，通过遵循此方案，您应该能够注射产生的AAV并随访其表达谱。并通过量化视网膜基因组DNA中的AAV基因组来提高转导效率。在这里，您可以看到数字液滴PCR的基本应用，您可以在其中对在视网膜细胞中转导的AAV基因组进行绝对定量。将注射的视网膜中的基因组DNA稀释到0.05%pluronic F 68溶液中，以达到每个样品每微升1纳克的终浓度。在20微升PCR反应混合物中进行液滴生成，包括一个纳克基因组DNA 125纳米摩尔引物和2X常绿超级混合物。将样品混合物加载到墨盒的中间部分以产生液滴。然后将70微升液滴生成油加入底排墨盒中。将垫圈放在墨盒顶部。确保垫圈和墨盒之间没有间隙。然后将其放入液滴发生器中。轻轻取顶部铃铛中产生的约40微升液滴。将液滴溶液加入半裙边96孔PCR反应板中。使用铝密封箔用PCR板密封器密封PCR板。将PCR板放入96孔热封热循环仪中。使用本文中指示的 ddPCR 协议。将PCR板放入液滴读数仪中以分析数据。观看此视频后，您应该能够执行基本的数字PCR技术。您也可以使用含有少量物质的受累细胞，这是数字PCR技术的理想选择。但是，应仔细调整目标DNA量，使其不超过数字液滴PCR的限值。以下是实验过程的流程图。我们进行了小规模的AAV生产，随后进行了AAV滴定。然后我们进行了玻璃体内注射。之后，我们通过眼底荧光成像量化了荧光强度。然后分离小鼠视网膜并从视网膜中提取基因组DNA用于ddPCR。滴定 AAV 对于正确量化 AAV 转导也至关重要。ddPCR依赖于稀释目标AAV基因的液滴的产生。具有代表性的ddPCR结果显示AVV基因组的阳性和阴性液滴。高于阈值的正数和负数低于阈值。在与稀释因子相乘后，发现AAV的滴度为每毫升基因组拷贝中的10至12。为了正确量化AAV转导效率，注射了几个视网膜。对注射的动物进行成像，并计算荧光平均强度。在两周的时间点，分离出视网膜。ddPCR是使用WPRE和18 S引物进行的。AAV注射视网膜的荧光强度与AAV基因组计算相关，显示了该方法的强大功能。在这里，我们展示了几种技术，包括小规模AAV制备玻璃体内注射。

Research

JoVE Journal

Neuroscience

In vivo Electroporation of Developing Mouse Retina

In vivo Electroporation of Developing Mouse Retina

在发展中国家小鼠视网膜体内电穿孔

The functional characterization of genes expressed during mammalian retinal development remains a significant challenge.  Gene targeting to generate ...

科研

神经科学

Assessment of Vascular Regeneration in the CNS Using the Mouse Retina

血管再生在中枢神经系统中使用的小鼠视网膜评估

The rodent retina is perhaps the most accessible mammalian system in which to investigate neurovascular interplay within the central nervous system (CNS). ...

Intracerebroventricular Viral Injection of the Neonatal Mouse Brain for Persistent and Widespread Neuronal Transduction

新生小鼠脑侧脑室注射病毒的持续和广泛的神经元传导

With the pace of scientific advancement accelerating rapidly, new methods are needed for experimental neuroscience to quickly and easily manipulate gene ...

Imaging Ca2+ Dynamics in Cone Photoreceptor Axon Terminals of the Mouse Retina

Imaging Ca2+ Dynamics in Cone Photoreceptor Axon Terminals of the Mouse Retina

成像钙<sup&gt; 2+</sup&gt;在小鼠视网膜锥光感受器轴突终末动态

Retinal cone photoreceptors (cones) serve daylight vision and are the basis of color discrimination. They are subject to degeneration, often leading to ...

Ocular Kinematics Measured by In Vitro Stimulation of the Cranial Nerves in the Turtle

Ocular Kinematics Measured by In Vitro Stimulation of the Cranial Nerves in the Turtle

体外刺激对龟脑神经的运动测量

After animals are euthanized, their tissues begin to die. Turtles offer an advantage because of a longer survival time of their tissues, especially when ...

A Volumetric Method for Quantification of Cerebral Vasospasm in a Murine Model of Subarachnoid Hemorrhage

小鼠蛛网膜下腔出血模型脑血管痉挛量化的容积法

Subarachnoid hemorrhage (SAH) is a subtype of hemorrhagic stroke. Cerebral vasospasm that occurs in the aftermath of the bleeding is an important factor ...

Transpupillary Two-Photon In Vivo Imaging of the Mouse Retina

小鼠视网膜的经瞳双光子体内成像

The retina transforms light signals from the environment into electrical signals that are propagated to the brain. Diseases of the retina are prevalent ...

Two Peeling Methods for the Isolation of Photoreceptor Cell Compartments in the Mouse Retina for Protein Analysis

两种剥离方法，用于分离小鼠视网膜中的光感受器细胞区室以进行蛋白质分析

Rod photoreceptors are highly polarized sensory neurons with distinct compartments. Mouse rods are long (~80 &#181;m) and thin (~2 &#181;m) and are ...

In Vivo Vascular Injury Readouts in Mouse Retina to Promote Reproducibility

In Vivo Vascular Injury Readouts in Mouse Retina to Promote Reproducibility

Advancements in ophthalmic imaging tools offer an unprecedented level of access to researchers working with animal models of neurovascular injury. To ...