Institute of Experimental Hematology, Hannover Medical School, Hannover, Germany.
Institute of Experimental Hematology, Hannover Medical School, Hannover, Germany.
Exp Hematol. 2014 Jul;42(7):505-515.e7. doi: 10.1016/j.exphem.2014.03.004. Epub 2014 Mar 24.
Tetracycline-regulated integrating vectors allow pharmacologically controlled genetic modification of murine and human hematopoietic stem cells and provide the opportunity for time- and dose-controlled reversible transgene expression in hematopoietic stem cells in vitro and in vivo. However, the background activity of tetracycline-regulated promoters (tetPs) in the absence of induction or vector integration in the vicinity of proto-oncogenes can diminish the advantages of the system. Here we investigated the effect of lentiviral transduction rate on tetP background activity, vector copy number (VCN), and clonal variability as a consequence of vector integration. We found an exponential relationship between VCN and gene transfer/expression level, accompanied by a linear relationship between VCN and tetP background activity. Long-term murine transplantation studies demonstrated stable and reversible transgene expression in serial recipients. Although analysis of associated clonal composition revealed development of clonal dominance in the presence and absence of induction, no indications of disease presented during the observation period. The majority of tetracycline-regulated vector integration sites were identified in intron/exons of metabolic/housekeeping and signaling genes or in noncoding/repeat regions of the genome. Furthermore, we demonstrated that the nature of the selected transgene might affect tetP background activity and inducibility in vivo. Limiting tetP-regulated gene transfer may avoid generation of clones with high VCN and enhanced tetP background activity. Our data help to establish physiologic and pathophysiologic systems to study dose-dependent mechanisms triggered by different levels of transgene expression in the context of basic HSC biology and cellular transformation models.
四环素调控的整合载体允许对鼠类和人类造血干细胞进行药理学控制的基因修饰，并为体外和体内造血干细胞中四环素调控启动子（tetPs）的时间和剂量控制的可逆转基因表达提供机会。然而，在没有诱导或载体整合的情况下，tetPs 的背景活性邻近原癌基因会降低该系统的优势。在这里，我们研究了慢病毒转导率对 tetP 背景活性、载体拷贝数（VCN）和载体整合后克隆变异性的影响。我们发现 VCN 与基因转移/表达水平之间存在指数关系，同时 VCN 与 tetP 背景活性之间存在线性关系。长期的鼠类移植研究表明，在连续受体中存在稳定和可逆的转基因表达。虽然对相关克隆组成的分析表明，在诱导存在或不存在的情况下会出现克隆优势，但在观察期间没有出现疾病迹象。四环素调控的载体整合位点大多数位于代谢/管家基因和信号基因的内含子/外显子中，或者在基因组的非编码/重复区域。此外，我们证明所选转基因的性质可能会影响体内 tetP 背景活性和诱导能力。限制 tetP 调控的基因转移可能会避免产生具有高 VCN 和增强的 tetP 背景活性的克隆。我们的数据有助于建立生理和病理生理系统，以研究在基本 HSC 生物学和细胞转化模型背景下，不同水平的转基因表达所触发的剂量依赖性机制。
