Microglia, the ever-changing resident immune cells of the central nervous system, have come under intense scrutiny for their role in Alzheimer’s disease and other neuroinflammatory conditions. Tracking these famously elusive cells just got easier, with new mouse models that allow researchers to color microglia in vivo, or to make genetic changes in them at will. Using precision gene editing, two groups have targeted the recently discovered microglia-specific Tmem119 gene to drive expression of green or red fluorescent protein markers, or Cre recombinase, solely in microglia.
- Scientists need tools to study microglia biology.
- New mouse lines use TMEM119 to label and control gene expression specifically in microglia.
- Some lines already are publicly available.
In one effort, Tobias Kaiser and Guoping Feng at MIT, Cambridge, Massachusetts, generated lines of mice whose microglia glow green, or express an inducible Cre gene. The second project, from Wassim Elyaman and colleagues at Columbia University Medical Center, New York, introduces mice with red microglia. Importantly, the approach distinguishes microglia from peripheral monocytes and macrophages, which do not express Tmem119. Both reports are currently online on bioRχiv.
The Kaiser and Feng mice are available for purchase from The Jackson Laboratory. Elyaman told Alzforum his mice are available to collaborators under a material transfer agreement.
Glowing Glia. Knock-in mice express marker protein or Cre along with Tmem119. (Left) P2A ribosome skipping peptide allows normal expression of Tmem119 plus transgenes from a single mRNA. (Right) Tamoxifen-inducible Cre-dependent expression of tdTomato occurs only in IBA1-expressing microglia. [Courtesy of Kaiser and Feng, 2019, bioRχiv.]
Researchers contacted by Alzforum expressed enthusiasm about the mice. Jaime Grutzendler, Yale University, New Haven, Connecticut, said he had ordered the MIT mice, and Marco Prinz, University of Freiburg, Germany, wrote that his lab has the MIT mice and researchers are busy checking them out, but have no data yet.
Tmem119 was only recently identified as an exclusive marker of human and mouse microglia (Bennett et al., 2016). A highly expressed transmembrane protein, it has quickly become the gold standard for immunohistochemical identification or purification of microglia. Tmem119’s function remains a mystery, but its expression levels, developmental regulation, and abundance all point to an important role in the cells’ physiology.
With that in mind, Kaiser and Feng wanted to avoid changing microglial Tmem119 expression while inserting reporter genes. “Our goal was to perturb the cells as little as possible,” Kaiser told Alzforum.
To that end, they used CRISPR/Cas9 to introduce genes for either enhanced green fluorescent protein (EGFP) or a tamoxifen-inducible Cre recombinase into the Tmem119 stop codon. They preceded this with a ribosome skipping signal, aiming for normal production of Tmem119, plus EGFP or Cre proteins from a single mRNA. As hoped, this engineering did not change TMEM119 mRNA or protein levels in the brain, and the microglia maintained their normal morphology.
What about specificity? In the EGFP mice, the scientists found essentially complete labeling of brain microglia, but no signal in oligodendrocytes, neurons or astrocytes, or in macrophages or blood monocytes. The inducible Cre reporter was slightly less specific, and did result in scattered expression of a Cre-dependent marker in other brain macrophages, in leptomeningeal cells, and blood monocytes. This was minimal, though, and at far lower levels than seen with commonly used Cx3Cr1 lines.
“This could be very useful in a variety of studies, including in AD and other diseases and injuries, where there is always the controversy as to whether the cells observed in the brain are all microglia, or also include peripherally derived cells,” Grutzendler said.
In the second report, co-author Samuel Hasson, then at Pfizer, used a similar CRISPR-targeting strategy and ribosome skipping peptide to place the tdTomato red fluorescence reporter just after the last exon of Tmem119, again causing no harm to TEME119 expression. Chunsheng Ruan in Elyaman’s group led the effort to characterize the mice, which revealed fluorescence in more than 95 percent of microglia in the brain and spinal cord, but not in other cells, and no expression in peripheral tissues. Even mice aged nearly 2 years maintained reporter expression. Because of its brightness, tdTomato is useful for in vivo imaging, and the scientists recorded movies of microglia streaming to a site of brain injury in living Tmeme119-tdTomato mice. Elyaman told Alzforum they are already at work crossing the animals with mouse models of AD and other diseases.—Pat McCaffrey
- Bennett ML, Bennett FC, Liddelow SA, Ajami B, Zamanian JL, Fernhoff NB, Mulinyawe SB, Bohlen CJ, Adil A, Tucker A, Weissman IL, Chang EF, Li G, Grant GA, Hayden Gephart MG, Barres BA. New tools for studying microglia in the mouse and human CNS. Proc Natl Acad Sci U S A. 2016 Mar 22;113(12):E1738-46. Epub 2016 Feb 16 PubMed.
No Available Further Reading
- Ruan C, Sun L, Kroshilina A, Beckers L, De Jager P, Bradshaw EM, Hasson SA, Yang G, Elyaman W. A novel Tmem119-tdTomato reporter mouse model for studying microglia in the central nervous system. Brain Behav Immun. 2020 Jan;83:180-191. Epub 2019 Oct 8 PubMed.
- Kaiser T, Feng G. Tmem119-EGFP and Tmem119-CreERT2 transgenic mice for labeling and manipulating microglia. bioRχiv. May 19, 2019