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Dose response and clonal variability of lentiviral tetracycline-regulated vectors in murine hematopoietic cells

Authors
  • Kustikova, Olga S.
  • Stahlhut, Maike
  • Ha, Teng-Cheong
  • Scherer, Ralph
  • Schambach, Axel
  • Baum, Christopher1, 2, 3, 2, 4, 5, 6
  • 1 Institute of Experimental Hematology
  • 2 Hannover Medical School
  • 3 Institute for Biometry
  • 4 Division of Hematology/Oncology
  • 5 Children's Hospital Boston
  • 6 Harvard Medical School
Type
Published Article
Journal
Experimental Hematology
Publisher
Elsevier
Publication Date
Jan 01, 2014
Accepted Date
Mar 06, 2014
Identifiers
DOI: 10.1016/j.exphem.2014.03.004
Source
Elsevier
Keywords
License
Unknown

Abstract

Tetracycline-regulated integrating vectors allow pharmacologically controlled genetic modification of murine and human hematopoietic stem cells (HSCs) and provide the opportunity for time- and dose-controlled reversible transgene expression in HSCs in vitro and in vivo. However, the background activity of tetracycline-regulated promoters (tetP) 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 within non-coding/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 be proposed to 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 HSCs biology and cellular transformation models.

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