Comparative clonal analysis of reconstitution kinetics after transplantation of hematopoietic stem cells gene marked with a lentiviral SIN or a γ-retroviral LTR vector
Comparative clonal analysis of reconstitution kinetics after transplantation of hematopoietic stem cells gene marked with a lentiviral SIN or a γ-retroviral LTR vector
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DOI：
10.1016/j.exphem.2012.09.003
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发表时间：
2013-01-01
影响因子：
2.6
通讯作者：
Fehse, Boris
中科院分区：
文献类型：
--
作者：
Cornils, Kerstin;Bartholomae, Cynthia C.;Fehse, Boris
逆转录病毒基因标记已成功用于临床前和临床移植环境。高灵敏度的载体插入位点测定技术，如线性扩增介导的聚合酶链反应（LAM-PCR）结合下一代测序，已被引入到评估基因标记的造血细胞在单细胞水平的组成。在这里，我们使用这些新的技术直接比较克隆重建动力学的小鼠移植骨髓来源的干细胞的遗传标记的标准，脾病灶形成病毒长末端重复序列（LTR）驱动的γ-逆转录病毒，或慢病毒自身灭活载体含有相同的，但内部脾病灶形成病毒衍生的增强子/启动子。我们观察到慢病毒自失活载体用于基因标记与更广泛的不同标记的造血克隆库相关。更重要的是，我们发现在随后的分析时间点，在脾病灶形成病毒LTR驱动的γ-逆转录病毒载体中，促进生长的克隆显性相关基因插入的概率显著更高。基于我们的数据，我们认为，LAM-PCR和下一代测序的结合使用代表了一个强有力的工具，用于分析克隆重建动力学的背景下，基因标记与整合载体。同时，我们的研究结果证明，使用多种限制性内切酶的LAM-PCR是必不可少的，以检测大多数或理想的所有个人干细胞克隆有助于造血。我们还发现，定量PCR等技术有助于回顾性分析单个造血干细胞克隆的重建动力学。最后，我们的研究结果证实了慢病毒自失活载体标记与γ-逆转录病毒LTR载体相比具有较低的遗传毒性风险的概念。(c)2013 ISEH -血液学和干细胞学会。爱思唯尔公司出版
Retroviral gene marking has been used successfully in preclinical and clinical transplantation settings. Highly sensitive techniques for vector insertion-site determination, such as linear amplification mediated polymerase chain reaction (LAM-PCR) in conjunction with next-generation sequencing, have been introduced to assess the composition of gene-marked hematopoiesis at a single-cell level. Here we used these novel techniques for directly comparing clonal reconstitution kinetics in mice transplanted with bone-marrow-derived stem cells genetically marked with either a standard, spleen focus-forming virus long terminal repeat (LTR)-driven gamma-retroviral, or a lentiviral self-inactivating vector containing an identical but internal spleen focus-forming virus-derived enhancer/promoter. We observed that the use of the lentiviral self-inactivating vector for gene marking was associated with a broader repertoire of differently marked hematopoietic clones. More importantly, we found a significantly higher probability of insertions in growth-promoting, clonal-dominance-associated genes in the spleen focus-forming virus LTR-driven gamma-retroviral vector at later time points of analysis. Based on our data, we suggest that the combined use of LAM-PCR and next-generation sequencing represents a potent tool for the analysis of clonal reconstitution kinetics in the context of gene marking with integrated vectors. At the same time, our findings prove that the use of multiple restriction enzymes for LAM-PCR is indispensable to detect most or ideally all individual stem cell clones contributing to hematopoiesis. We have also found that techniques such as quantitative PCR can be helpful to retrospectively analyze reconstitution kinetics for individual hematopoietic stem cell clones. Finally, our results confirm the notion that marking with lentiviral self-inactivating vectors is associated with a lower risk of genotoxicity as compared with gamma-retroviral LTR vectors. (c) 2013 ISEH - Society for Hematology and Stem Cells. Published by Elsevier Inc.