title: Dual-promoter lentiviral system allows inducible expression of noxious proteins in macrophages.
abstract: In-depth studies of innate immunity require efficient genetic manipulation of macrophages, which is especially difficult in primary macrophages. We have developed a lentiviral system for inducible gene expression both in macrophage cell lines and in primary macrophages. A transgenic mouse strain C3H.TgN(SRA-rtTA) that expresses reverse tetracycline transactivator (rtTA) under the control of macrophage-specific promoter, a modified human Scavenger Receptor A (SRA) promoter was generated. For gene delivery, we constructed a dual-promoter lentiviral vector, in which expression of a "gene-of-interest" is driven by a doxycycline-inducible promoter and the expression of a selectable surface marker is driven by an independent constitutive promoter UBC. This vector is used for transduction of bone marrow-derived macrophage precursors. The transduced cells can be enriched to 95-99% purity using marker-specific monoclonal antibodies, expanded and differentiated into mature macrophages or myeloid dendritic cells. We also successfully used this approach for inducible protein expression in hard to transfect macrophage cell lines. Because many proteins, which are expressed by activated or infected macrophages, possess cytotoxic, anti-proliferative or pro-apoptotic activities, generation of stable macrophage cell lines that constitutively express those proteins is impossible. Our method will be especially useful to study immunity-related macrophage proteins in their physiological context during macrophage activation or infection.
authors.affiliation: Department of Immunology and Infectious Diseases, Harvard School of Public Health, 667 Huntington Avenue, Boston, MA 02115, USA.
authors.forename: Hui
authors.initials: H
authors.lastname: Pan
authors.forename: Gustavo
authors.initials: G
authors.lastname: Mostoslavsky
authors.forename: Evgeny
authors.initials: E
authors.lastname: Eruslanov
authors.forename: Darrell N
authors.initials: DN
authors.lastname: Kotton
authors.forename: Igor
authors.initials: I
authors.lastname: Kramnik
mesh_terms: D000818:Animals; D002454:Cell Differentiation; D049109:Cell Proliferation; D002478:Cells, Cultured; D003587:Cytomegalovirus / Q000235:genetics; D004318:Doxycycline / Q000494:pharmacology; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins / Q000096:biosynthesis; D006412:Hematopoietic Stem Cells / Q000378:metabolism*; D006801:Humans; D007371:Interferon-gamma / Q000378:metabolism; D016086:Lentivirus / Q000187:drug effects / Q000235:genetics*; D008264:Macrophages / Q000378:metabolism*; D051379:Mice; D008809:Mice, Inbred C3H; D008822:Mice, Transgenic; D041681:NIH 3T3 Cells; D011401:Promoter Regions, Genetic / Q000187:drug effects; D011485:Protein Binding; D039104:RNA 3' Polyadenylation Signals; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D051117:Scavenger Receptors, Class A / Q000235:genetics; D013539:Simian virus 40 / Q000235:genetics; D013997:Time Factors; D015534:Trans-Activators / Q000096:biosynthesis* / Q000235:genetics; D014161:Transduction, Genetic; D025822:Ubiquitin C / Q000235:genetics
chemical_list: C465035:Ifi75 protein, mouse; D011993:Recombinant Fusion Proteins; D051117:Scavenger Receptors, Class A; D015534:Trans-Activators; D025822:Ubiquitin C; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D007371:Interferon-gamma; D004318:Doxycycline