Comparative analysis of CreER transgenic mice for the study of brain macrophages: A case study

Chappell‐Maor, Louise; Kolesnikov, Masha; Kim, Jung Seok; Shemer, Anat; Haimon, Zhana; Grozovski, Jonathan; Boura‐Halfon, Sigalit; Masuda, Takahiro; Prinz, Marco; Jung, Steffen

doi:10.1002/eji.201948342

Cited by 51 publications

(38 citation statements)

References 50 publications

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“…In the second study, Chappell‐Maor et al. not only confirm reporter leakiness as described in point 2 above, but also reiterate another concern with Cre/loxP systems; nonspecific and/or undesired targeting [16]. With some exceptions, Cre/CreER mouse strains are rarely uniquely specific to a single cell‐type.…”

Section: Figurementioning

confidence: 65%

“…Previous articles have cast concerns on conditional mutagenesis, particularly the accuracy and efficiency of excision of different floxed alleles [11–14]. Expanding on this, this issue of the European Journal of Immunology features two elegantly designed and complementing studies, which provide an insight into the complexity of inducible gene targeting [15,16]. Both studies made use of two tamoxifen‐inducible CreER strains commonly used for manipulating brain macrophages; Cx3cr1 CreER and Sall1 CreER .…”

Section: Figurementioning

confidence: 99%

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STOP floxing around: Specificity and leakiness of inducible Cre/loxP systems

Stifter

Greter

2020

Eur J Immunol

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Cre and CreER mouse strains are powerful tools that have proven invaluable for investigating the function of genes and for the fate‐mapping of cell populations. The fidelity of these systems however are becoming more and more contested. In this issue of the European Journal of Immunology, Van Hove et al. and Chappell‐Maor et al. carefully dissect the cellular specificities of two commonly used CreER mouse strains for the study of CNS macrophages; Cx3cr1CreER and Sall1CreER. Both studies elegantly highlight that CreER strains, as well as the "floxed" allele to be targeted, need to be carefully selected and properly characterized in order to ensure reproducible and robust data and interpretations. These studies are a cautionary tale for this technology, but also highlight that we must continuously question and improve our experimental approaches.

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Section: Figurementioning

confidence: 65%

Section: Figurementioning

confidence: 99%

STOP floxing around: Specificity and leakiness of inducible Cre/loxP systems

Stifter

Greter

2020

Eur J Immunol

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“…The same holds true for the Cx3cr1 CreERT2 strain, since it has been recently shown that Cx3cr1 Cre can drive rearrangements in neurons, most likely transiently during development . We also refer to the recent comprehensive analysis by the Jung group which describes complementary findings and shows via the RiboTag approach that Sall1 CreERT2 induces recombination in neurons and other glia . While the Cx3cr1 CreERT2 line did not induce recombination of the RiboTag allele in neurons , we cannot exclude leaky neuronal recombination for all other loxP flanked sequences, which will depend on the nature and size of the floxed allele.…”

mentioning

confidence: 68%

“…We also refer to the recent comprehensive analysis by the Jung group which describes complementary findings and shows via the RiboTag approach that Sall1 CreERT2 induces recombination in neurons and other glia . While the Cx3cr1 CreERT2 line did not induce recombination of the RiboTag allele in neurons , we cannot exclude leaky neuronal recombination for all other loxP flanked sequences, which will depend on the nature and size of the floxed allele. Finally, when using the Cx3cr1 CreERT2 strain to dissect the role of tissue‐resident versus recruited myeloid cells that infiltrate the brain during disease , it is advisable to confirm that leaky recombination is low in the latter population.…”

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confidence: 82%

Identifying the variables that drive tamoxifen‐independent CreERT2 recombination: Implications for microglial fate mapping and gene deletions

et al. 2019

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Ligand‐dependent Cre recombinases such as the CreERT2 system allow for tamoxifen‐inducible Cre recombination. Important examples are the Cx3cr1‐CreERT2 and Sall1‐CreERT2 lines that are widely used for fate mapping and gene deletion studies of brain macrophages. Our results now show that both CreERT2 lines can exhibit a high rate of tamoxifen‐independent “leaky” excision with some reporter strains, while this is not observed with others. We suggest that this disparity is determined by the length of the floxed transcriptional STOP cassette that is incorporated in the various reporter lines. In addition, the rate of spontaneous recombination was also determined by the CreERT2 expression levels and the longevity of the CreERT2‐expressing cells. The implications of these results are discussed in the context of fate mapping and inducible gene deletion studies in macrophages and microglia.

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“…Aside from Cx3CR1, knock-in GFP and CreER lines have been generated for the microglial-enriched transcription factor Sall1 (Buttgereit et al, 2016). However, significant caveats for these lines exist, including the fact that Sall1-GFP and CreER both disrupt the function of the Sall1 gene, and that Sall1-CreER marks a broad range of non-myeloid CNS cell types (Chappell-Maor et al, 2019).…”

Section: Discussionmentioning

confidence: 99%

New Tools for Genetically Targeting Myeloid Populations in the Central Nervous System

Gl¹,

Lizama²,

Ae³

et al. 2019

Preprint

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As the resident macrophages of the brain and spinal cord, microglia are crucial for the phagocytosis of infectious agents, apoptotic cells and synapses. Developmentally, microglia originate from the embryonic yolk sac and serve important roles in the sculpting of neonatal neural circuits. During brain injury or infection, bone-marrow derived macrophages invade neural tissue, making it difficult to distinguish between invading macrophages and resident microglia. In addition to circulation-derived monocytes, other non-microglial central nervous system (CNS) macrophage subtypes include borderzone (meningeal and perivascular) and choroid plexus macrophages. To distinguish between resident microglia and these other CNS macrophage subtypes, we generated a P2ry12-CreER mouse line. P2RY12 is a microglialspecific nucleotide sensing GPCR that is important for microglial response to tissue damage. Using immunofluorescent labeling and flow cytometry experiments, we show that P2ry12-CreER recombination is exceptionally specific to parenchymal microglia. We also perform ribosome immunoprecipitations and transcriptional profiling of P2ry12-CreER recombined cells, using a Cre-dependent Rpl22-HA mouse line. By identifying genes enriched in this dataset that are not correspondingly enriched in a broader Cx3CR1-CreER; Rpl22 dataset, we isolate a number of borderzone macrophage-specific transcripts, including the gene PF4. Using a PF4-Cre mouse line, we show that PF4 expression robustly marks borderzone macrophages. Together, we demonstrate two new methods to genetically target distinct CNS macrophage subtypes.

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Comparative analysis of CreER transgenic mice for the study of brain macrophages: A case study

Cited by 51 publications

References 50 publications

STOP floxing around: Specificity and leakiness of inducible Cre/loxP systems

STOP floxing around: Specificity and leakiness of inducible Cre/loxP systems

Identifying the variables that drive tamoxifen‐independent CreERT2 recombination: Implications for microglial fate mapping and gene deletions

New Tools for Genetically Targeting Myeloid Populations in the Central Nervous System

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