Objective: Macrophages are critical components of diverse microenvironments (ME) in adulthood, as well as during embryogenesis. Their role in development precludes the use of gene-targeting and knockout approaches for studying their function. Hence, we proposed to create a macrophage-specific inducible transgenic mouse where genes can be turned on or off at will.
Materials and methods: A transgenic mouse in which the reverse tetracycline activator (rtTA-M2) is expressed under the hCD68 promoter for macrophage-specific gene induction was developed and crossed with a second transgenic reporter mouse strain in which the gene for green fluorescent protein (GFP) is under the control of tetracycline responsive element promoter. After doxycycline induction of the double transgenic animals (designated CD68-rtTA-tet-GFP), inducible expression of GFP was characterized by multicolor flow cytometric analysis of blood, marrow, and spleen cells and by demonstration of GFP expression in fresh-frozen sections in diverse tissues.
Results: In bone marrow, inducible GFP expression was not confined to, or inclusive of, all cells expressing the classical macrophage markers, such as F4/80. However, GFP-expressing cells in thioglycollate-elicited peritoneal macrophages were also positive for F4/80 and monocyte-macrophage-specific 2 antigen. Interestingly, flow analysis also indicated little overlap between the F4/80 and CSF-1R-positive populations. Fresh-frozen samples of tissues known to contain macrophages revealed GFP-expressing cells with variable morphologies.
Conclusion: Our results show that the hCD68 promoter directs gene expression in a macrophage population distinct from that defined by classical monocyte-macrophage markers or promoters. Whether this population is functionally distinct remains to be established.