A Novel Tmem119-tdTomato Reporter Mouse Model for Studying Microglia in the Central Nervous System

Abstract

Microglia are resident immune cells of the central nervous system (CNS). The exact role of microglia in the physiopathology of CNS disorders is not clear due to lack of tools to discriminate between CNS resident and infiltrated innate immune cells. Here, we present a novel reporter mouse model targeting a microglia-specific marker (TMEM119) for studying the function of microglia in health and disease. By placing a reporter cassette (GSG-3xFlag-P2A-tdTomato) between the coding sequence of exon 2 and 3UTR of the Tmem119 gene using CRISPR/Cas9 technology, we generated a Tmem119-tdTomato knock-in mouse strain. Gene expression assay showed no difference of endogenous Tmem119 mRNA level in the CNS of Tmem119tdTomato/+ relative to control Wild-type mice. The cells expressing tdTomato-were recognized by immunofluorescence staining using commercially available anti-TMEM119 antibodies. Using immunofluorescence and flow cytometry techniques, tdTomato+ cells were detected throughout the CNS, but not in peripheral tissues of adult Tmem119tdTomato/+ mice. In addition, aging does not seem to influence TMEM119 expression as tdTomato+ cells were detectable in the CNS of older mice (300 and 540 days old). Further immunofluorescence characterization shows that the tdTomato+ cells were highly colocalized with Iba1+ cells (microglia and macrophages) in the brain, but not with NeuN- (neurons), GFAP- (astrocytes) or Olig2- (oligodendrocytes) labeled cells. Moreover, flow cytometry analysis of brain tissues of adult mice demonstrates that the majority of microglial CD45lowCD11b+ cells (96.6%) are tdTomato positive. Functionally, using a laser-induced injury model, we measured time-lapse activation of tdTomato-labeled microglia by transcranial two-photon microscopy in live Tmem119tdTomato/+ mice. Taken together, the Tmem119-tdTomato reporter mouse model will serve as a valuable tool to specifically study the role of microglia in health and disease.