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Multimodal lentiviral vectors (LVs) allow switching between constitutive and tetracycline-regulated gene co-expressions in genetically modified cells. Transduction of murine primary hematopoietic progenitor cells (HPCs) with multimodal LVs in the absence of doxycycline ensures the constitutive expression of gene of interest 1 (GOI1) only. In the presence of doxycycline, induced tetracycline-regulated expression of a second GOI (GOI2) allows evaluation of the collaboration between two genes. Drug removal retains constitutive expression, which allows the contribution of an individual gene into created networks to be studied. Doxycycline-dependent switching can be tracked via fluorescent markers coupled to constitutive and tetracycline-regulated GOIs. This article describes transduction of murine primary HPCs with different doses of multimodal LVs, distinct cytokine conditions, and their influence on the number and viability of cells co-expressing both collaborating GOIs upon doxycycline induction. A 2-week protocol is provided for multimodal LV production, titer determination, and evaluation of tetracycline responsive promoter background activity in a murine fibroblast cell line. The power of this model to assess the dose/time/order-controlled contribution of single and multiple genes into hematopoietic networks opens new routes in reprogramming, stem cell, and leukemia biology.

human gene therapy, part b. methods/human gene therapy. part b. methods

Multimodal Lentiviral Vectors for Pharmacologically Controlled Switching Between Constitutive Single Gene Expression and Tetracycline-Regulated Multiple Gene Collaboration