High-accuracy biodistribution analysis of adeno-associated virus variants by double barcode sequencing
High-accuracy biodistribution analysis of adeno-associated virus variants by double barcode sequencing
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DOI：
10.1038/mtm.2015.41
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发表时间：
2015-01-01
影响因子：
4.7
通讯作者：
Zolotukhin, Sergei
中科院分区：
文献类型：
--
作者：
Marsic, Damien;Mendez-Gomez, Hector R.;Zolotukhin, Sergei
生物分布分析是评估腺相关病毒（AAV）衣壳变异的关键步骤，无论是自然分离的还是通过合理设计或定向进化产生的。事实上，在筛选候选载体时，准确了解哪些组织被感染以及感染效率是至关重要的。我们描述了一种新的载体pTR-UF50-BC的设计、验证和应用，该载体编码生物发光蛋白、荧光蛋白和DNA条形码，可用于在生物体、器官、组织或细胞水平上可视化转导的定位。此外，通过将衣壳变体连接到载体的不同条形码版本，并使用条形码引物扩增来自各种组织样本的条形码区域，可以高精度和高效地分析病毒基因组的生物分布。
Biodistribution analysis is a key step in the evaluation of adeno-associated virus (AAV) capsid variants, whether natural isolates or produced by rational design or directed evolution. Indeed, when screening candidate vectors, accurate knowledge about which tissues are infected and how efficiently is essential. We describe the design, validation, and application of a new vector, pTR-UF50-BC, encoding a bioluminescent protein, a fluorescent protein and a DNA barcode, which can be used to visualize localization of transduction at the organism, organ, tissue, or cellular levels. In addition, by linking capsid variants to different barcoded versions of the vector and amplifying the barcode region from various tissue samples using barcoded primers, biodistribution of viral genomes can be analyzed with high accuracy and efficiency.