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Depletion and Reconstitution of Macrophages in Mice
Published on: August 1, 2012
Stromelysin-1 (MMP-3) expression driven by a macrophage-specific promoter results in reduced viability in transgenic
R P Fabunmi1, K J Moore, P Libby
1Lipid Metabolism Unit, Massachusetts General Hospital, GRJ 1328, 55 Fruit Street, Boston, MA 02114, USA.
Abstract:
Macrophage expression of matrix degrading metalloproteinases (MMPs) in human atheroma has been found to occur in rupture-prone areas of plaques. To investigate the effect of metalloproteinase activity on plaque stability, we attempted to generate mice that expressed a stromelysin-1 (MMP-3) transgene specifically in macrophages. Promoter sequences taken from a macrophage-tropic lentivirus (visna) were used to drive transgene expression. The transgene construct was expressed in macrophages in vitro and its autoactivation was established by casein zymography. Transgenic mice generated with this construct died at or before birth. No gross anatomical changes were observed in these mice. Embryos arising from a second round of oocyte injections with the transgene were examined at day 16 of gestation. Of the products of conception, approximately 40% resulted in vacant conceptuses. Only one animal of 38 examined carried the transgene and its expression of MMP-3 mRNA at E16 was faintly detected by RT-PCR. When a non-toxic reporter gene, luciferase, was substituted for the MMP-3 cDNA, healthy transgenic mice were produced that expressed the reporter gene in a wide variety of tissue macrophages, including those located in the brain, testis, lung, and thymus. These studies suggest that constitutive expression of MMP-3 in diverse populations of tissue macrophages leads to prenatal or neonatal death in the mouse. It appears likely that more sophisticated transcriptional control of MMP-3 expression will be required in order to generate stromelysin-1 transgenic mice that could be useful models for studying overexpression of this metalloproteinase's activity in the lesional macrophages of atherosclerotic plaques.
Insights
Generating mice with macrophage-specific stromelysin-1 (MMP-3) expression led to prenatal death, suggesting MMP-3 overexpression is detrimental to development. Further research requires refined control over MMP-3 gene expression in macrophages.
Area of Science:
- Cardiovascular Biology
- Genetics and Transgenics
- Molecular Medicine
Background:
- Macrophage-derived matrix metalloproteinases (MMPs), particularly stromelysin-1 (MMP-3), are implicated in atherosclerotic plaque instability.
- Understanding the role of MMP-3 in plaque rupture requires models that allow for specific manipulation of its expression in macrophages.
Purpose of the Study:
- To investigate the impact of metalloproteinase activity on plaque stability by generating transgenic mice with macrophage-specific MMP-3 expression.
- To establish a suitable mouse model for studying the effects of MMP-3 overexpression in the context of atherosclerosis.
Main Methods:
- Utilized macrophage-tropic lentivirus promoter sequences to drive MMP-3 transgene expression specifically in macrophages.
- Generated transgenic mice and assessed transgene expression and viability.
- Employed casein zymography for MMP-3 autoactivation assessment and RT-PCR for MMP-3 mRNA detection.
- Used a luciferase reporter gene to validate macrophage-specific expression in a separate cohort.
Main Results:
- Transgenic mice with the MMP-3 construct exhibited embryonic lethality, dying at or before birth.
- Approximately 40% of conceptuses were vacant, and only one of 38 examined animals carried the transgene with faint MMP-3 mRNA expression.
- Transgenic mice expressing a luciferase reporter gene in macrophages were viable and healthy, indicating successful macrophage-specific gene delivery.
Conclusions:
- Constitutive expression of MMP-3 in diverse tissue macrophages results in prenatal or neonatal death in mice.
- Sophisticated transcriptional control strategies are necessary to develop functional MMP-3 transgenic mouse models for studying atherosclerosis.
