Generation of retroviruses for the overexpression of cytosolic and mitochondrial glutathione reductase in macrophages in vivo

Abstract

Retroviral gene transfer and bone marrow transplantation has been used by many investigators to study the role of macrophage proteins in different mouse models of human disease. While this approach is faster and less expensive than generating transgenic mice with macrophage-specific promoters and applicable to a wider array of mouse models, it has been hampered by two major drawbacks: labor-intensive cloning procedures involved in generating retroviral vectors for each gene of interest and low viral titers. Here we describe the construction of a MSCV-based retroviral vector that can serve as an acceptor vector for commercially available Cre-lox-compatible donor vectors. Using this new retroviral vector in combination with a FACS approach to enhance viral titers, we generated high-titer retroviruses carrying either EGFP-tagged cytosolic or EGFP-tagged mitochondria-targeted glutathione reductase. We show that the introduction of these constructs via retroviral gene transfer and bone marrow transplantation into atherosclerosis-prone LDL receptor-null mice results in the long-term increase in macrophage glutathione reductase activity.

Fig. 1

Restriction maps for pMSCV-GR-EGFP, pMSCV-MTS-GR-EGFP and pMSCV EGFP vectors: Relevant restriction sites are indicated

Fig. 2

Retrovirus-mediated expression of EGFP, GR-EGFP and MTS-GR-EGFP in Phoenix Eco cells: Cells grown on cover slips in 12-well plates were collected 48 h after transfection, fixed and mounted as described under “Methods”. Cells expressing EGFP (A, D), GR-EGFP (B, D) and MTS-GR-EGFP (C, F) are shown. Panels (A, B, C) represent Hoffmann-contrast images of panels D, E and F, respectively (200×)

Fig. 3

Glutathione reductase activity in peritoneal macrophages isolated from LDL-R^−/− mice reconstituted with EGFP, GR-EGFP or MTS-GR-EGFP-expressing bone marrow: Bone marrow cells were transduced with retroviral vectors carrying either EGFP, GR-EGFP or MTS-GR-EGFP and transplanted into lethally irradiated LDLR^−/− mice. Fifteen weeks after bone marrow transplantation, peritoneal macrophages were isolated from the recipient mice and cellular GR activity was determined as described under “Methods”