title: Lentiviral vector system for coordinated constitutive and drug controlled tetracycline-regulated gene co-expression.
abstract: Constitutive co-expression of cooperating transgenes using retroviral integrating vectors is frequently used for genetic modification of different cell types to establish therapeutic or cancer models. However, such approaches are unable to dissect the influence of dose, order and reversibility of transgene expression on the fate of newly developed therapeutic/malignant phenotypes. We present a modular lentiviral vector system, which provides expression of constitutive and inducible components. To demonstrate its functionality, we constitutively expressed the well-described transcription factor Meis1 followed by inducible co-expression of collaborating partner Hoxa9 under the control of tetracycline responsive promoters in murine fibroblasts and primary hematopoietic progenitor cells (HPCs). Fluorescent markers to track transgene co-expression revealed tightly controlled, efficiently inducible and reversible but cell type dependent gene transfer over time. We demonstrated dose-dependent blockade of myeloid differentiation when both Meis1/Hoxa9 were concomitantly overexpressed in primary HPCs in vitro, but the absence of the transformed phenotype in non-induced samples or when Hoxa9 expression was down-regulated. This system combines the advantages of lentiviral gene transfer and the opportunity for drug-controlled co-expression of multiple transgenes to dissect, among others, gene networks governing complex cell behavior, such as proto-oncogene dose-dependent leukemogenic pathways or collaborating mechanisms of genes enhancing competitive fitness of hematopoietic cells.
authors.affiliation: Institute of Experimental Hematology, Hannover Medical School, Hannover, Germany.
authors.forename: Maike
authors.initials: M
authors.lastname: Stahlhut
authors.affiliation: Institute of Experimental Hematology, Hannover Medical School, Hannover, Germany; Department of Hematology, Hemostasis, Oncology and Stem Cell Transplantation, Hannover Medical School, Hannover, Germany.
authors.forename: Adrian
authors.initials: A
authors.lastname: Schwarzer
authors.affiliation: Department of Hematology, Hemostasis, Oncology and Stem Cell Transplantation, Hannover Medical School, Hannover, Germany.
authors.forename: Matthias
authors.initials: M
authors.lastname: Eder
authors.affiliation: Institute of Experimental Hematology, Hannover Medical School, Hannover, Germany; Department of Hematology, Hemostasis, Oncology and Stem Cell Transplantation, Hannover Medical School, Hannover, Germany.
authors.forename: Min
authors.initials: M
authors.lastname: Yang
authors.affiliation: Institute of Experimental Hematology, Hannover Medical School, Hannover, Germany; Department of Hematology, Hemostasis, Oncology and Stem Cell Transplantation, Hannover Medical School, Hannover, Germany.
authors.forename: Zhixiong
authors.initials: Z
authors.lastname: Li
authors.affiliation: Institute of Experimental Hematology, Hannover Medical School, Hannover, Germany.
authors.forename: Michael
authors.initials: M
authors.lastname: Morgan
authors.affiliation: Institute of Experimental Hematology, Hannover Medical School, Hannover, Germany; Division of Hematology/Oncology, Boston Children's Hospital, Harvard Medical School, Boston, USA.
authors.forename: Axel
authors.initials: A
authors.lastname: Schambach
authors.affiliation: Institute of Experimental Hematology, Hannover Medical School, Hannover, Germany. Electronic address: Kustikova.Olga@mh-hannover.de.
authors.forename: Olga S
authors.initials: OS
authors.lastname: Kustikova
mesh_terms: D000818:Animals; D002454:Cell Differentiation; D002460:Cell Line; D002478:Cells, Cultured; D005786:Gene Expression Regulation / Q000187:drug effects; D005822:Genetic Vectors / Q000235:genetics*; D006412:Hematopoietic Stem Cells / Q000166:cytology / Q000378:metabolism; D018398:Homeodomain Proteins / Q000235:genetics*; D006801:Humans; D016086:Lentivirus / Q000235:genetics*; D051379:Mice; D008810:Mice, Inbred C57BL; D022423:Myeloid Cells / Q000166:cytology / Q000378:metabolism; D000075064:Myeloid Ecotropic Viral Integration Site 1 Protein; D009363:Neoplasm Proteins / Q000235:genetics*; D011401:Promoter Regions, Genetic / Q000187:drug effects; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology*; D000090063:Proto-Oncogene Mas; D013752:Tetracycline / Q000494:pharmacology*; D014161:Transduction, Genetic; D019076:Transgenes / Q000187:drug effects
chemical_list: D018398:Homeodomain Proteins; C000715470:MAS1 protein, human; C000616391:MEIS1 protein, human; C000616392:Meis1 protein, mouse; D000075064:Myeloid Ecotropic Viral Integration Site 1 Protein; D009363:Neoplasm Proteins; D011500:Protein Synthesis Inhibitors; D000090063:Proto-Oncogene Mas; C097899:homeobox protein HOXA9; D013752:Tetracycline
keywords: Constitutive; Dose-dependent; Fate decision; Genetic modification of HSCs; Lentiviral; Multiple transgenes; Tetracycline-regulated