Harkey Michael A, Kaul Rajinder, Jacobs Michael A, Kurre Peter, Bovee Don, Levy Ruth, Blau C Anthony
Department of Transplantation Biology, Fred Hutchinson Cancer Research Center, Seattle, WA 98109-1024, USA.
Stem Cells Dev. 2007 Jun;16(3):381-92. doi: 10.1089/scd.2007.0015.
Retroviral integration provides a unique and heritable genomic tag for a target cell and its progeny, enabling studies of clonal composition and repopulation kinetics after gene transfer into hematopoietic stem cells. The clonal tracking method, linear amplification-mediated polymerase chain reaction (LAM-PCR) is widely employed to follow the hematopoietic output of retrovirally marked stem cells. Here we examine the capabilities and limitations of conventional LAM-PCR to track individual clones in a complex multiclonal mix. Using artificial mixtures of retrovirally marked, single-cell-derived clones, we demonstrate that LAM-PCR fails to detect 30-40% of the clones, even after exhaustive analysis. Furthermore, the relative abundance of specific clones within a mix is not accurately represented, deviating by as much as 60-fold from their true abundance. We describe an optimized, multiarm, high-throughput modification of LAM-PCR that improves the global detection capacity to greater than 90% with exhaustive sampling, facilitates accurate estimates of the total pool size from smaller samplings, and provides a rapid, cost-effective approach to the generation of large insertion-site data bases required for evaluation of vector integration preferences. The inability to estimate the abundance of individual clones within mixtures remains a serious limitation. Thus, although LAM-PCR is a powerful tool for identification of integration sites and for estimations of clonal complexity, it fails to provide the semiquantitative information necessary for direct, reliable tracking of individual clones in a chimeric background.
逆转录病毒整合为靶细胞及其子代提供了一个独特且可遗传的基因组标签，使得在基因导入造血干细胞后能够对克隆组成和再增殖动力学进行研究。克隆追踪方法，即线性扩增介导的聚合酶链反应（LAM-PCR）被广泛用于追踪逆转录病毒标记的干细胞的造血输出。在此，我们研究了传统LAM-PCR在复杂多克隆混合物中追踪单个克隆的能力和局限性。使用逆转录病毒标记的单细胞来源克隆的人工混合物，我们证明即使经过详尽分析，LAM-PCR仍无法检测到30%-40%的克隆。此外，混合物中特定克隆的相对丰度没有得到准确呈现，与它们的真实丰度偏差高达60倍。我们描述了一种优化的、多臂的、高通量的LAM-PCR改进方法，该方法通过详尽采样将全局检测能力提高到90%以上，便于从小样本中准确估计总库大小，并为生成评估载体整合偏好所需的大型插入位点数据库提供了一种快速、经济高效的方法。无法估计混合物中单个克隆的丰度仍然是一个严重的局限性。因此，尽管LAM-PCR是用于鉴定整合位点和估计克隆复杂性的强大工具，但它无法提供在嵌合背景中直接、可靠地追踪单个克隆所需的半定量信息。
