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

Comments

  1. Since Mariko Bennett and Ben Barres published Tmem119 as a marker of microglia distinct from peripheral myeloid cells in 2016, the field has been abuzz with the ability to discern between peripheral immune cells and resident microglia (Bennett et al., 2016). While for many applications it is possible that such distinctions are not important (for instance in disease responses in which there is a similar contribution of both microglia and, say, macrophages), for many developmental and brain-specific immune responses, having such a marker has been imperative.

    The advent of these new Tmem119-targeted eGFP and CreERT2 mouse lines provide two key tools for the field. That the eGFP line completely and faithfully labels microglia at different time points of development, and that this fluorescence is stable for FACS purification, will no doubt see the investigation of live-cell imaging and future sequencing experiments beyond what is currently possible (and easily accessible for many labs). One could cross these lines with peripheral immune cell tdTomato reporter lines to determine the CNS migration differences or changes in the integration with disease pathologies in live animals—very exciting. Similarly, the CreERT2 line provides inducible specificity to manipulate a whole host of microglia-specific functions, and is going to enable one to tease the specific functions of resident versus infiltrating immune cells—an exciting prospect to be sure.

    The highlights of the mouse include: no change in morphology, gene expression, or numbers in eGFP/CreERT2 lines, high specificity for Tmem119+ CNS-resident microglia, with no expression in other non-microglia monophagocytic cells, e.g., meningeal macrophages. There is some early expression of Tmem119 in non-microglial endothelial cells, but this is itself a feature, not a bug, as it allows the study of microglia and endothelial cells until postnatal day 1 (P1) and microglia specifically after P3 and later.

    To highlight how excited the field is, a similar set of lines is being produced by Mariko Bennett, and upon seeing the publication from Kaiser and Feng, her Twitter response was as follows: “‘Scooped’ and can’t even be grumpy because it’s gonna be an awesome resource and is already available at Jax. We’ll be sure to compare the lines as soon as we’re sure ours is ready for prime time.” It will be exciting to cross the Tmem119CreERT2 line with ribotag, bac- and nuc-TRAP lines to further investigations into microglia-specific biology in health and disease.

    References:

    . 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.

    View all comments by Shane Liddelow
  2. We ordered the mice for trial in our lab, but have not received them yet. In principle they sound super useful, as they have not knocked out the endogenous Tmem119 gene. This is in contrast to other lines available, such as the CX3CR1, which is widely used but has a loss of function of CX3CR1. LOF of CX3CR1 clearly poses problems for many experiments, as even partial loss of CX3CR1 has significant phenotypes.

    The other potentially interesting thing about these mice is that they appear to preferentially target microglia but not peripheral monocytes. 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 some peripherally derived cells.

    In summary, for us these mice may prove to be highly useful. We are hoping that the GFP brightness in the TMEM119 is large so that we can do live imaging.

    View all comments by Jaime Grutzendler
  3. These look useful and I wouldn’t be surprised if many groups took advantage. The current state of the art for this type of approach is an analogous pair of Cx3cr1-driven mice, which have the drawback, as the authors indicate, of also labeling other non-microglia myeloid populations. We used these for a data set included in our Cell Reports manuscript from last year. This new pair of nice looks pretty clean, based on the figures presented (Cd11b:Cd45-high unlabeled in FACS and CD163 meningeal macrophages unlabeled in IHC). If we had used the Tmem119::EGFP mice instead, then I expect our data set would have been even more specific.

    View all comments by Brad Friedman

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References

Paper Citations

  1. . 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.

Further Reading

No Available Further Reading

Primary Papers

  1. . A Novel Tmem119-tdTomato Reporter Mouse Model for Studying Microglia in the Central Nervous System. bioRχiv, June 10, 2019
  2. . Tmem119-EGFP and Tmem119-CreERT2 transgenic mice for labeling and manipulating microglia. bioRχiv. May 19, 2019