A comparative analysis of microglial inducible Cre lines

Faust, Travis E.; Feinberg, Philip A; O’Connor, Ciara; Kawaguchi, Riki; Chan, Andrew; Strasburger, Hayley J.; Masuda, Takahiro; Amann, Lukas; Knobeloch, Klaus-Peter E; Prinz, Marco; Schaefer, Anne; Schafer, Dorothy P.

doi:10.1101/2023.01.09.523268

Cited by 17 publications

(32 citation statements)

References 33 publications

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“…1B,C ). Furthermore, in the absence of tamoxifen, we did not find evidence of recombination (tdTomato labelled microglia), consistent with a recent study 46 .…”

Section: Resultssupporting

confidence: 93%

Sex and interferon gamma signaling regulate microglia migration in adult mouse cortexin vivo

Boghozian

Sharma

Cheema

et al. 2023

Preprint

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Although microglia possess the unique ability to migrate, whether mobility is evident in all microglia, is sex dependent, and what molecular mechanisms drive this, is poorly understood in the adult brain. Using longitudinal in vivo imaging of sparsely labelled microglia, we find a relatively small population of microglia are mobile under normal conditions. Following injury (microbleed), the population of mobile microglia increased in a sex-dependent manner, with male microglia migrating significantly greater distances towards the microbleed relative to their female counterparts. To understand the signaling pathways involved, we interrogated the role of interferon gamma (IFNγ). Our data show that in male mice, stimulating microglia with IFNγ or inducible microglial specific knockdown of IFNγ receptor 1 stimulates or inhibits migration, respectively, whereas female microglia were generally unaffected. These findings highlight the diversity of microglia migratory responses to injury, its dependence on sex and the signaling mechanisms that modulate this behavior.

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“…1B,C ). Furthermore, in the absence of tamoxifen, we did not find evidence of recombination (tdTomato labelled microglia), consistent with a recent study 46 .…”

Section: Resultssupporting

confidence: 93%

Sex and interferon gamma signaling regulate microglia migration in adult mouse cortexin vivo

Boghozian

Sharma

Cheema

et al. 2023

Preprint

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“…With our rigorous and comprehensive analysis of the four commercially available microglia-CreER mouse lines on the five key features we chose to focus on, we identify important caveats and strengths for these lines which will be of broad significance for researchers interested in performing conditional gene deletion in microglia. While we are preparing for this manuscript, a preprint of an independent study evaluating microglial CreER lines was reported 40 , including evaluation of HexB CreER mice. Our two studies provide both overlapping and distinct recommendations and guidelines for the use of these powerful tools.…”

Section: Discussionmentioning

confidence: 99%

Finding the right tool: a comprehensive evaluation of microglial inducible cre mouse models

Bedolla

McKinsey

Ware

et al. 2023

Preprint

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The recent proliferation of new Cre and CreER recombinase lines provides researchers with a diverse toolkit to study microglial gene function. To determine how best to apply these lines in studies of microglial gene function, a thorough and detailed comparison of their properties is needed. Here, we examined four different microglial CreER lines (Cx3cr1CreER(Litt), Cx3cr1CreER(Jung), P2ry12CreER, Tmem119CreER), focusing on (1) recombination specificity; (2) leakiness - degree of non-tamoxifen recombination in microglia and other cells; (3) efficiency of tamoxifen-induced recombination; (4) extra-neural recombination -the degree of recombination in cells outside the CNS, particularly myelo/monocyte lineages (5) off-target effects in the context of neonatal brain development. We identify important caveats and strengths for these lines which will provide broad significance for researchers interested in performing conditional gene deletion in microglia. We also provide data emphasizing the potential of these lines for injury models that result in the recruitment of splenic immune cells.

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“…Previous carefully designed work using inducible microglia CreERT2 lines showed varying degrees of leakiness in the context of sensitive reporters and high CreERT2 expression levels (Chappell-Maor et al, 2020; Van Hove et al, 2020). Conversely, inefficient recombination has been observed in the context of less sensitive reporters and lower Cre expression levels (Alicia Bedolla et al, 2023; Travis E. Faust et al, 2023). To test the generalizability of loxP recombination by the Fcrls-2A-Cre line, we crossed Fcrls-2A-Cre mice to three different reporters of differential sensitivity and genomic context (Figure 3A-C).…”

Section: Resultsmentioning

confidence: 99%

“…Conversely, inefficient recombination has been observed in the context of less sensitive reporters and lower Cre expression levels (Alicia Bedolla et al, 2023;Travis E. Faust et al, 2023). To test the generalizability of loxP recombination by the Fcrls-2A-Cre line, we crossed Fcrls-2A-Cre mice to three different reporters of differential sensitivity and genomic context (Figure 3A-C).…”

Section: Fcrls-2a-cre Mice Effectively Recombine Floxed Dna In All Mi...mentioning

confidence: 99%

Transgenic Targeting ofFcrlsCreates a Highly Efficient Constitutively Active Microglia Cre Line with Differentiated Specificity

Kaiser,

Dattero,

et al. 2023

Preprint

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Microglia carry out important functions as the resident macrophages of the brain. To study their role in health and disease, the research community needs tools to genetically modify them with maximum completeness in a manner that distinguishes them from closely related cell-types, such as monocytes. While currently available tamoxifen-inducible CreERT2 lines are able to achieve the differentiation from other cells, the field needs improved and publicly available constitutively active Cre lines, especially ones with favorable efficiency and specificity profiles for studies where high recombination efficiency is imperative and where tamoxifen administration is contraindicated. Here, we leverage the microglia-specificFcrlsgene to generate mice expressing Cre. Using genomic methods, we show correct positioning of the transgene and intact microglia homeostasis inFcrls-2A-Cremice. CrossingFcrls-2A-Cremice to four different reporters, we demonstrate highly efficient recombination in microglia across differentially sensitive loxP alleles in different genomic contexts, indicating robust applicability of the line. Further, we show that microglia recombine a loxP reporter during early embryonic development, supporting the use of the line for developmental studies. Finally, using immunofluorescence and flow cytometry, we reveal that most border associated macrophages (BAMs) are also targeted whereas only few liver and spleen macrophages and virtually no white blood cell subsets exhibit Cre activity, distinguishing this line from another publicly available Cre line,Cx3cr1-CreM(MMRRC).Fcrls-2A-Cremice are immediately available (JAX Stock #036591) and serve as a valuable addition to the community’s microglia toolbox by providing highly efficient constitutive Cre activity with excellent specificity, particularly for studies where tamoxifen administration is undesirable.Significance StatementThe microglia toolbox is continuously growing with more transgenic lines and most recently even viral tools becoming available. When selecting a Cre driver line, investigators must weigh relative strengths and weaknesses of available lines and carefully make the best choice for their given application. These tradeoffs include (1) availability and ease of employment, (2) chromosomal positioning of Cre with respect to the floxed allele (should not be on the same chromosome for conditional knockout studies), (3) activity level of a given Cre line and thus completeness of recombination across the microglia population, (4) specificity with respect to acceptable off-target cell types and tissues, (5) temporal aspects including earliest onset of Cre expression or inducibility, (6) robustness in disease contexts, and (7) potential perturbation of microglia homeostasis through Cre itself or disruption of the targeting locus. When selecting a mouse line, it is evident that there may not be a one-size-fits all solution but an application-based preference and choice from the diverse repertoire of microglia tools.Fcrls-2A-Cremice are an excellent addition to this toolbox.

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A comparative analysis of microglial inducible Cre lines

Cited by 17 publications

References 33 publications

Sex and interferon gamma signaling regulate microglia migration in adult mouse cortexin vivo

Sex and interferon gamma signaling regulate microglia migration in adult mouse cortexin vivo

Finding the right tool: a comprehensive evaluation of microglial inducible cre mouse models

Transgenic Targeting ofFcrlsCreates a Highly Efficient Constitutively Active Microglia Cre Line with Differentiated Specificity

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