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Efficient technique for immortalization of murine microglial cells relevant for studies in murine models of multiple
Julie K Olson1, Scott S Zamvil, Stephen D Miller
1Department of Microbiology-Immunology and Interdepartmental Immunobiology Program, Northwestern University Medical School, 303 East Chicago Avenue, Chicago, IL 60611, USA.
Abstract:
Microglia are macrophage-like cells that populate the central nervous system (CNS) and become activated upon injury or infection. Microglia have been implicated as playing critical roles in various CNS diseases including multiple sclerosis (MS), a human autoimmune demyelinating disease, as well as in other neurodegenerative diseases. Two well-characterized models of MS, relapsing experimental autoimmune encephalomyelitis (R-EAE) and Theiler's murine encephalomyelitis virus (TMEV)-induced demyelinating disease, are inducible in SJL mice and model the relapsing-remitting and chronic-progressive forms of MS, respectively. These models are useful for the study of the mechanisms of initiation, progression, and therapy of the disease. Currently, a major limitation to studying the functions of microglia in these murine models of MS is the restricted number of cells capable of being isolated from the CNS of neonatal mice and propagated in culture. The current studies describe the preparation of SV-40 large T antigen-immortalized mouse microglia lines, M4T.4 and M4T.6, from the SJL/J mice. The immortalization technique was very efficient requiring only 6 weeks to develop long-term, highly replicating cell lines. The resulting microglia cell lines remain quiescent, but are induced to express various immune cytokines and to function as efficient antigen presenting cells upon activation with IFN-gamma or infection with TMEV. Thus, the SV-40 large T antigen immortalized microglia lines react to innate and infectious stimuli similar to primary microglia isolated from neonatal mice, but are more easily maintained in culture. This technique should allow for the efficient cultivation of large numbers of microglial cells from a variety of disease-relevant mouse strains, including knock-out and transgenic mice.
Insights
Researchers developed immortalized mouse microglia cell lines, M4T.4 and M4T.6, for studying central nervous system diseases like multiple sclerosis. These cell lines mimic primary microglia responses, aiding research into neuroinflammation and disease mechanisms.
Area of Science:
- Neuroscience
- Immunology
- Cell Biology
Background:
- Microglia are key immune cells in the central nervous system (CNS), implicated in neurodegenerative diseases like multiple sclerosis (MS).
- Studying microglia in MS mouse models is limited by the difficulty of isolating and culturing sufficient primary cells from neonatal mice.
- Existing MS models, such as experimental autoimmune encephalomyelitis (EAE) and Theiler's murine encephalomyelitis virus (TMEV) infection, are crucial for understanding disease progression and therapeutic strategies.
Purpose of the Study:
- To establish immortalized mouse microglia cell lines for enhanced research into CNS diseases.
- To overcome the limitations of primary microglia culture in studying microglial function in MS models.
- To create a reproducible and scalable method for microglia research.
Main Methods:
- Utilized SV-40 large T antigen to immortalize microglia from SJL/J mice, creating M4T.4 and M4T.6 cell lines.
- Assessed the efficiency and time required for cell line development (approximately 6 weeks).
- Evaluated the functional characteristics of immortalized microglia, including cytokine expression and antigen presentation upon activation.
Main Results:
- Successfully generated long-term, highly replicating immortalized mouse microglia cell lines (M4T.4 and M4T.6).
- These cell lines remain quiescent but can be activated to express immune cytokines and function as antigen-presenting cells (APCs) when stimulated with IFN-gamma or TMEV.
- The immortalized microglia exhibit responses similar to primary microglia but are significantly easier to maintain in culture.
Conclusions:
- SV-40 large T antigen immortalization provides an efficient method to create robust microglia cell lines from various mouse strains.
- These immortalized microglia lines offer a valuable tool for studying microglial roles in CNS diseases, including MS, by enabling large-scale cultivation.
- This technique facilitates research using genetically modified (knock-out, transgenic) mice relevant to disease pathology.