期刊
JOURNAL OF IMMUNOLOGICAL METHODS
卷 329, 期 1-2, 页码 31-44出版社
ELSEVIER SCIENCE BV
DOI: 10.1016/j.jim.2007.09.009
关键词
lentiviral vectors; macrophage; tetracycline-inducible expression; Ipr1 gene; interferon-activation; protein-protein interaction
资金
- NHLBI NIH HHS [R01 HL059836-10, R01 HL059836] Funding Source: Medline
- NIAID NIH HHS [R01 AI049421, AI49421, R01 AI049421-05, P01 AI056296-010003, P01 AI056296] Funding Source: Medline
- NATIONAL HEART, LUNG, AND BLOOD INSTITUTE [R01HL059836] Funding Source: NIH RePORTER
- NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES [P01AI056296, R01AI049421] Funding Source: NIH RePORTER
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 proapoptotic 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. (C) 2007 Elsevier B.V. All rights reserved.
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