Department of Cell Biology, Section Stem Cell Biology, University Medical Center Groningen, University of Groningen, Groningen, The Netherlands.
Blood. 2010 Apr 1;115(13):2610-8. doi: 10.1182/blood-2009-06-229757. Epub 2010 Jan 21.
Clonal analysis is important for many areas of hematopoietic stem cell research, including in vitro cell expansion, gene therapy, and cancer progression and treatment. A common approach to measure clonality of retrovirally transduced cells is to perform integration site analysis using Southern blotting or polymerase chain reaction-based methods. Although these methods are useful in principle, they generally provide a low-resolution, biased, and incomplete assessment of clonality. To overcome those limitations, we labeled retroviral vectors with random sequence tags or "barcodes." On integration, each vector introduces a unique, identifiable, and heritable mark into the host cell genome, allowing the clonal progeny of each cell to be tracked over time. By coupling the barcoding method to a sequencing-based detection system, we could identify major and minor clones in 2 distinct cell culture systems in vitro and in a long-term transplantation setting. In addition, we demonstrate how clonal analysis can be complemented with transgene expression and integration site analysis. This cellular barcoding tool permits a simple, sensitive assessment of clonality and holds great promise for future gene therapy protocols in humans, and any other applications when clonal tracking is important.
克隆分析对于造血干细胞研究的许多领域都很重要，包括体外细胞扩增、基因治疗以及癌症的发生和治疗。一种常用的测量逆转录病毒转导细胞克隆性的方法是使用 Southern 印迹或聚合酶链反应（PCR）方法进行整合位点分析。虽然这些方法在理论上是有用的，但它们通常提供低分辨率、有偏差和不完整的克隆性评估。为了克服这些限制，我们使用随机序列标签或“条形码”标记逆转录病毒载体。在整合过程中，每个载体将独特的、可识别的和可遗传的标记引入宿主细胞基因组中，从而可以随着时间的推移追踪每个细胞的克隆后代。通过将条形码方法与基于测序的检测系统相结合，我们可以在体外的 2 种不同细胞培养系统和长期移植环境中识别主要和次要克隆。此外，我们还展示了如何通过转导基因表达和整合位点分析来补充克隆分析。这种细胞条形码工具可简单、灵敏地评估克隆性，并有望在未来的人类基因治疗方案以及任何其他需要克隆追踪的应用中得到广泛应用。
