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 (SR-A) 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.

Amelioration of emphysema in mice through lentiviral transduction of long-lived pulmonary alveolar macrophages

“…Although macrophages have been considered difficult to efficiently transfect either in vitro or in vivo (6,32), we demonstrate that VSV-G-pseudotyped lentiviral vectors efficiently transduced AMs, which persisted for at least 2 years in the airspaces of mouse lung.…”

“…An alternative approach is the transduction of differentiated cells. However, some differentiated cell types, such as macrophages, are difficult to transduce (6). Furthermore, terminally differentiated cells often have a short turnover time and are replaced by precursor cells, causing rapid loss of transgene expression.…”

Deletion of the murine scavenger receptor CD68

“…Moreover, expression appeared to correlate with macrophage activation ( 5,6 ). Consistent with these observations, the CD68 promoter has been exploited successfully to direct the expression of transgenes toward the macrophage lineage in vivo (7)(8)(9). dendritic cells (DC) were recovered ( ‫ف‬ 75% CD11c + ) and replated for an additional two or three days prior to analysis.…”

Dominant Role of the sst1 Locus in Pathogenesis of Necrotizing Lung Granulomas during Chronic Tuberculosis Infection and Reactivation in Genetically Resistant Hosts

“…We found that overexpression of the full-length Ipr1 transgene in the sst1 S macrophages increases their resistance to necrotic cell death induced by the virulent intracellular pathogens M. tuberculosis and Listeria monocytogenes in vitro. The Ipr1 is an IFN-inducible protein localized in macrophage nuclei, where it participates in formation of protein complexes in an IFNinducible manner 36 and associates with chromatin (Pan, in preparation). Because it has several protein interaction domains but no known functional domains, Ipr1 is likely an adaptor protein that plays a currently unrecognized role in macrophage responsiveness to type I and II IFNs, and, possibly, adaptation to harsh environments encountered by macrophages within inflammatory lesions.…”