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Lentiviral vector system for coordinated constitutive and drug controlled tetracycline-regulated gene co-expression
Maike Stahlhut1, Adrian Schwarzer2, Matthias Eder3
1Institute of Experimental Hematology, Hannover Medical School, Hannover, Germany.
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.
Insights
This study introduces a novel lentiviral vector system for controlled gene expression. The system allows researchers to study gene networks and dose-dependent effects in cell differentiation and transformation.
Area of Science:
- Molecular Biology
- Gene Therapy
- Cell Biology
Background:
- Retroviral vectors enable constitutive transgene co-expression for cell modification.
- Current methods lack control over transgene expression's dose, order, and reversibility.
- Dissecting gene networks requires precise control over multiple transgene expression.
Purpose of the Study:
- To develop a modular lentiviral vector system for constitutive and inducible transgene co-expression.
- To investigate the role of Meis1 and Hoxa9 in myeloid differentiation and transformation.
- To demonstrate the system's utility in studying gene networks and dose-dependent effects.
Main Methods:
- Developed a modular lentiviral vector system with constitutive and inducible components.
- Utilized tetracycline-responsive promoters for drug-controlled gene expression.
- Co-expressed Meis1 (constitutive) and Hoxa9 (inducible) in murine fibroblasts and hematopoietic progenitor cells (HPCs).
- Employed fluorescent markers to track transgene co-expression and gene transfer efficiency.
Main Results:
- Achieved tightly controlled, inducible, and reversible transgene co-expression, dependent on cell type.
- Demonstrated dose-dependent blockade of myeloid differentiation in HPCs upon Meis1/Hoxa9 co-expression.
- Observed no transformed phenotype in non-induced or Hoxa9-downregulated samples.
- Confirmed efficient and cell type-dependent gene transfer over time.
Conclusions:
- The developed lentiviral vector system offers precise control over multiple transgene expression.
- This system enables the dissection of gene networks, including proto-oncogene dose-dependent leukemogenesis.
- It facilitates the study of collaborating gene mechanisms in hematopoietic cell fitness and transformation.
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