Generation of retroviruses for the overexpression of cytosolic and mitochondrial glutathione reductase in macrophages in vivo
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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.
KeywordsRetrovirus Gene Transfer Glutathione Reductase Macrophage Atherosclerosis
We would like to thank Jill Chelowa and Li Xu for their technical assistance. This work was supported by grants to R.A. from the National Institutes of Heath (HL-70963) and the American Heart Association (455176B).