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

Summary 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.


Introduction
Microglia, the resident macrophages of the neural parenchyma, regulate a variety of processes necessary for brain development, homeostatic function, and injury/disease response. In addition to their role in innate immunity in the brain and surveillance of parenchyma, during both development and adulthood, microglia have been credited with synaptic pruning which plays a vital role in synaptic plasticity [1][2][3][4] . Outside the realms of development and homeostasis, microglia also respond to disease and injury, with varying activation profiles depending on the challenge 5,6 . With the increase in knowledge and recognition of these processes has come a significant expansion in the number of Cre and CreER recombinase mouse lines to enable precise genetic targeting of microglia [7][8][9][10] . Manipulations of the fractalkine receptor Cx3cr1 gene locus have been one of the primary methods used to drive Cre and CreER expression in microglia (reviewed in 11 ). Indeed, Cx3cr1 is strongly expressed in microglia as well as other myeloid subsets, providing relatively specific gene targeting. Since their establishment, these lines have been collectively utilized in over 1000 published reports. These reports highlight the broad utility of Cx3cr1-based gene targeting but also describe several potentially important drawbacks including non-microglia recombination (lack of specificity) 12 , leakiness 13 , and off-target effects 12 . Based on then-emerging transcriptomic profiling, Sall1 CreER mice 14 were proposed to be more specific for the genetic manipulation of microglial cells than Cx3cr1 CreER mice. However, a detailed characterization later found that Sall1 CreER recombines neural-ectodermal lineages including neurons, astrocytes and oligodendrocyte populations, and also has significant tamoxifen-independent (leaky) recombination in microglia 12 . Furthermore, Sall1 CreER , like Cx3cr1 CreER mice, was generated as a knock-in/knock-out at the endogenous gene locus. As Sall1 is known to be critical for the maintenance of microglial homeostasis and activation 14,15 , heterozygous loss of Sall1 in -CreER mice could have important impacts on microglia complicating interpretation of lineage tracing and gene knockout experiments.
In recent years, several new CreER mice were generated to overcome the drawbacks of existing microglia-targeting lines. In particular, Tmem119 CreER and P2ry12 CreER lines were made to have increased specificity for microglia while sparing brain border macrophages, such as perivascular and pial macrophages. In addition to this increased specificity, these new lines lack recombination in circulating monocytes, making it easier to distinguish microglia from invading Cx3cr1 CreER(Litt) (generated from the Littman lab 16 ), Cx3cr1 CreER(Jung) (generated from the Jung lab 17 ), Tmem119 CreER 10 , and P2ry12 CreER 8 . We report that there is significant variability across lines in their leakiness and in their ability to recombine floxed alleles which we show is related both to loxP distance and intrinsic CreER activity or expression. We describe our unsuccessful efforts to improve recombination efficiency by crossing to the new iSuReCre mouse line. We also document significant differences in splenic recombination in the studied CreER lines, reflective of potential injury-induced monocyte recruitment from the spleen. In total, these comparative analyses provide important data for research groups eager to determine whether a particular microglial recombining CreER line would be appropriate and useful for studies of cell lineage tracing and/or conditional gene mutation in microglia.

Different degrees of leakiness in microglia CreER lines:
We first looked to confirm microglia specificity while also assessing leakiness in P2ry12 CreER and Tmem119 CreER mice. We analyzed Cx3cr1 CreER(Jung) and Cx3cr1 CreER(Litt) , both previously well characterized, as baseline controls. For these studies, we took advantage of what is widely regarded as the most sensitive Cre-recombinase reporter, Ai9/Ai14, and a less sensitive Cre reporter, ROSA26-YFP (hereafter referred to as R26-YFP) for comparison. Note that because Cx3cr1 CreER(Litt) line carries an internal ribosome entry site (IRES) -enhanced yellow fluorescent protein (EYFP) gene reporter, we only examined a cross with the Ai9-tdTomato reporter in this line as Cx3cr1 CreER(Litt) Ai9. We assessed mice treated with tamoxifen (180mg/kg daily gavage for 5 days), compared to mice treated with vehicle (sunflower oil + ethanol) kept in separate cages throughout the induction and analysis period (to ensure no tamoxifen contamination). Vehicle treatment provides a control to tamoxifen treatment and also allows for an assessment of leakiness -the number and types of cells recombined in the absence of tamoxifen.
As expected, tamoxifen induction in adult Cx3cr1 CreER (Jung and Littman) mice resulted in recombination of the Ai9 tdTomato Cre reporter allele in microglia as well as brain border macrophages in the choroid plexus, meninges, and perivascular spaces (Fig 1 bottom, Fig S1). In contrast, tamoxifen treated Tmem119 CreER and P2ry12 CreER mice had more specific tdTomato labeling of microglia and no apparent labeling of brain border macrophages (Fig S1)  This finding was confirmed using FACS analysis ( Fig S2). We attribute this difference in Cre reporting to the intrinsic sensitivity of the two Cre recombinase reporters; a function of inter-loxP distances (0.9 kb in Ai9; 2.7 kb in R26-YFP), mRNA stabilizing elements (Ai9 has WRPE), enhanced promoter elements (Ai9 uses a strong CAG promoter), and native fluorescence intensity (Ai9 tdT vs YFP).
Using the Ai9 Cre-reporter, we similarly observed relatively high degrees of tamoxifenindependent recombination in microglia in the two Cx3cr1 CreER lines (82% and 28%, respectively), and sparse recombination (tdTomato expression) of microglia in P2ry12 CreER Ai9 and Tmem119 CreER Ai9 mice in the absence of tamoxifen (Fig 1, top). Similar to the P2ry12 CreER Ai9 mice, we observed sparse non-tamoxifen recombination of microglia in Tmem119 CreER Ai9 mice.
Using the R26-YFP reporter line, we observe much less tamoxifen-independent expression of the YFP reporter in the Cx3cr1 CreER(Jung) R26-YFP brain and observed almost no YFP+ cells in Tmem119 CreER or P2RY12 CreER R26-YFP mice (occasionally one YFP + cell in the whole brain section). Taken together, these data confirm the previously characterized microglia-specific recombination Tmem119 CreER and P2ry12 CreER , and document low rates of tamoxifen-independent Cre recombination (leakiness) in both of these lines as well as both Cx3cr1 CreER lines, with the greatest relative leakiness observed in the Cx3cr1 CreER(Litt) line.
Gene targeting efficiency is related both to intrinsic CreER expression/activity and inter-loxP target length.
Our data above show that the Tmem119 CreER and P2ry12 CreER lines are able to achieve highly efficient recombination in the Ai9 allele (95% and 96%, respectively), whereas the recombination efficiency in the R26-YFP allele is comparatively less (34% and 37%, Fig 1 bottom). Similarly, the rates of tamoxifen-independent recombination in the Cx3cr1 CreER(Jung) line is lower with the Ai9 recombined cells on a single cell basis using flow cytometry and immunohistochemistry (Fig 2).  Fig 2). These results suggest that although the shorter length floxed Ai9 reporter allele has a higher probability of recombination on a populational level, the recombination of the two different alleles in the same genomic loci is independently regulated; in any single cell, which allele is recombined is independent from the recombination of the alternate allele and does not always follow the size rule. Similarly, we speculate that recombination of a single copy of reporter allele does not always guarantee the recombination of both floxed alleles of any given target gene, especially when using a floxed reporter allele that has a short floxed region such as Ai9 or Ai14.
To more directly investigate the efficiency of microglial recombination, we generated different combinations of Cx3Cr1 CreER(Jung) (apparent "strong" Cre) or P2ry12 CreER (apparent "weak" Cre), bred to the following floxed alleles with varying inter-loxP distances (bred to homozygosity): Tgfb1 fl/fl(ex3) (< 0.5kb loxp distance), Alk5 fl/fl(ex3) (1.6-1.7 kb loxP distance). Mice were bred with a recombination reporter (R26-YFP or Ai9/Ai14) to facilitate isolation of microglia cells that have undergone at least one recombination event. Total RNA was extracted from sorted microglia cells in all mouse lines, followed by cDNA library preparation and quantitative realtime PCR using allele-specific forward and reverse primers spanning the floxed and neighboring exon.
Absence of gene amplification therefore indicates deletion of floxed exons.
Our results (Fig 3) show that the Cx3Cr1 CreER(Jung) line leads to significant and almost complete loss of the floxed exon in total RNA content for both Cx3cr1 CreER(Jung) Tgfb1 fl/fl and Cx3cr1 CreER(Jung) Alk5 fl/fl mice (Fig 3,  Because P2ry12 creER recombines ~ 30% of the total microglia cells at the R26-YFP locus, and our . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted April 17, 2023. sorting strategy enriches for YFP + cells, this efficiency rate is likely overestimating true recombination efficiency at the gene of interest. Indeed, in P2ry12 CreER Alk5 fl/fl -Ai9 (with the easier to recombine Ai9 reporter), >95 % of sorted IBA1 + microglia were tdT + , while Alk5 mRNA is 84% of WT (Fig 3), indicating a lower overall recombination efficiency in P2ry12 CreER mice compared to the CX3cr1 CreER(Jung) line. Taken together, our data suggest that both intrinsic CreER activities as well as target inter-loxP distance are important determinants in gene excision. Our results also highlight critical differences in apparent gene targeting efficiency when using various reporters, specific assessment of allele-specific gene loss, or methods to isolate/analyze a conditional gene targeting experiment (e.g. FACS versus immuno-histology).

Lack of iSuReCre recombination by P2ry12CreER.
Given the relatively weak recombination efficiency in P2ry12 CreER , we were interested in whether we might enhance recombination while maintaining cellular specificity. The new iSuReCre mouse line, with Cre-inducible expression of both Cre-recombinase and membranous (Mb) Tomato reporter (Fig 4a), was made to accomplish this exact goal 20 . We generated P2ry12 CreER iSuReCre mice, treated them with TAM at 4-8 weeks of age, then harvested and analyzed brains 2-4 weeks later. Our results (Fig 4) show that addition of the iSuReCre allele failed to label any microglia with the MbTomato reporter in the P2ry12 CreER line. Interestingly, there was sparse MbTomato labeling of neurons in the cortex and striatum of P2ry12 CreER iSuReCre mice which did not receive tamoxifen (Fig 4b-i), likely representing ectopic TAM-independent and cre-independent expression of MbTomato from the iSuReCre locus since we never observed neuronal recombination in P2ry12 CreER R26-YFP or P2ry12 CreER Ai9 mice. To examine whether the iSuRe mouse model works properly in other non-microglia brain-specific Cre drivers, we generated a DCX CreER iSuReCre mice as a positive control (Fig 4j). DCX CreER mice target immature DCX + neuroblasts in the dentate gyrus which later develop into mature NeuN + neurons 4 weeks after administration of TAM. Indeed, brains from DCX creER iSuReCre mice had strong MbTomato expression in DCX + neuroblasts and mature NeuN + /DCX (-) neurons at 5days (Fig 4k-n) and 4 weeks (Fig 4o-r) after TAM administration, respectively. These data confirm that the iSuReCre allele is recombined by DCX CreER , similar to other Cre and CreER lines tested in the original publication 20 , but is not recombined in adult microglia by P2ry12 CreER .
Persistent recombination of splenic macrophages in microglia CreER lines.
. CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made One potential downside to using Cx3cr1 CreER -based strategies in lineage tracking and conditional mutagenesis experiments is these lines' known recombination of circulating blood cells, and the potential for these cells to invade and take residence in the CNS. With this in mind, strategies were developed to specifically label brain microglia vs peripheral monocytes/macrophage followed by a waiting period of > 3 weeks after TAM administration to allow for the turnover of circulating cells by non-recombined Cx3cr1-negative bone marrow progenitors 21,22 . However, these strategies are cumbersome or not feasible in developmental and injury contexts in which recombination and engraftment occurs more rapidly than the 3 week "washout period" and might include cells derived from non-myeloid (non-marrow) sources. The spleen is a particularly important site for the storage and rapid deployment of monocytes in the setting of development and inflammation [23][24][25] and can be used as a surrogate for recombination circulating monocytes.
To this end, we examined the expression of recombination reporter genes in the spleens (matched to brain recombination, Fig 1) 4 weeks after TAM or vehicle treatment ( Fig 5). In Cx3cr1 CreER(Litt) Ai9 mice, we observed substantial tamoxifen-independent base level tdTomato and YFP reporter expression in IBA1 + splenic macrophages, and a strong increase in reporter recombination/expression with persistence of these cells 4 weeks after tamoxifen administration, mirroring tamoxifen-independent and -dependent reporter recombination in the brain (Fig 1). We observed recombination of splenic macrophages in P2ry12 CreER Ai9 mice comparable to Cx3cr1 CreER lines (as was previously reported), but less recombination of splenic macrophages in Tmem119 CreER mice (Fg5h-m). Interestingly, there was strong recombination of non-myeloid (IBA1-negative) cells which, by comparison to the brain, may represent splenic adventitial fibroblasts 26 . These data highlight a relatively high degree of recombination in non-microglial splenic macrophages in TAM-treated (and untreated) Cx3cr1 CreER and P2ry12 CreER mice, representing a previously un-explored reservoir of recombined cells that could replace microglia and complicate the interpretation of lineage tracing and gene deletion experiments using these lines.
Off target effects of tamoxifen induced microglia-CreER expression.
A recent evaluation of the Cx3cr1 CreER(Litt) line found that neonatal tamoxifen exposure resulted in microglia with reduced expression of homeostatic genes, and reciprocal induction of activation genes related, in part, to interferon signaling 27 . Notably, Tmem119 CreER did not show this same Cre-mediated non-specific effect in early postnatal microglia, and administration of tamoxifen to . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted April 17, 2023. ; https://doi.org/10.1101/2023.04.17.536878 doi: bioRxiv preprint adult Cx3cr1 CreER(Litt) mice showed no major phenotype. Given the high efficiency of Cx3Cr1 CreER mouse lines in targeting microglia on a populational level and likely still popular usage in future microglia gene knockout studies, we looked to determine whether this same phenotype existed in other microglia-targeting CreER lines.
Cx3cr1 CreER + (Jung and Littman), and P2ry12 CreER + mice were intercrossed with wild type mice, and newborn pups were given tamoxifen (50ug dose IG or 500ug dose IP) on postnatal day P1,2,3 or P4,5,6 and then brains were harvested and analyzed on P15 or P30 (Fig 6). For convenience we used brains from mice with either a Tgfbr2 fl/wt or Tgfb1 fl/wt(ex1) allele already in our colonies. In neonatally-induced Cx3cr1 CreER(Litt) mice, we observed wide-spread loss of the homeostatic marker P2ry12 in IBA1 + microglia, accompanied by their adoption of an activated morphology (Fig 6a). In contrast, and similar to the published report 27 , tamoxifen-administration to Cx3cr1 CreER(Litt) mice after P21 had no apparent effect on microglia homeostasis or activation In investigating the microglia phenotype observed in Cx3cr1 CreER(Litt) mice, we were struck by its similarity to microglia deficient in TGFβ-signaling (Itgb8;nestinCre and Tgfbr2 fl/fl ;Cx3cr1 CreER 29 , Tgfb1 -/-30 ; Tgfbr2 fl/fl Sall1 CreER 31 ; Lrrc33/Nrros -/-15 ), including loss of homeostatic signature genes such as P2ry12, HexB, SiglecH and Tmem119 with reciprocal upregulation of MgND/DAM (neurodegeneration-associated phenotype by microglia/diseaseassociated microglia) signature genes including ApoE, Axl, and Lglas3; and genes directly or indirectly related to interferon signaling including Irf7, Siglec1, and Mx1 (Fig 6a). Indeed, the transcriptional phenotype of microglia from neonatal (P15) Tgfbr2 fl/fl ;Cx3cr1 CreER mice are moderately correlated to those from neonatal tamoxifen-treated Cx3cr1 CreER(Litt) mice (R=0.56; P>2e-16). Importantly, these various TGF-β-signaling deficient mouse models were not generated using the Cx3cr1 CreER(Litt) line, raising the hypothesis that neonatal tamoxifen administration to Cx3cr1 CreER(Litt) mice might somehow dysregulate TGF-β signaling. We directly tested this by . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted April 17, 2023. ; https://doi.org/10.1101/2023.04.17.536878 doi: bioRxiv preprint immunostaining mouse brains for phosphorylated (p)Smad3, an indicator of canonical (SMADmediated) TGF-β signaling. While there was a trend for reduced pSmad3 immuno-fluorescent staining in microglia from TAM-treated compared to non-TAM treated controls, the overall change on the population level was not significantly different. These results suggest that reduced TGF-β1-signaling is not likely a primary driver of the microglial phenotype in Cx3cr1 CreER(Litt) mice, but could be a downstream consequence of increased INF-signaling as proposed by Suhasrabuddhe et al. Consistent with this, INF and TGF-β1/Smad signaling are co-regulated in activated microglia 32 , and Smad2/3 is known to directly regulate the expression of several homeostatic markers including P2ry12 33 .

Discussion
The use of the Cre-LoxP system has revolutionized biological research by enabling celltype-specific gene manipulation. The addition of temporal control to Cre activity was introduced by the addition of a modified estrogen receptor (ER) ligand binding domain 34 providing exquisite precision in targeting and tracking ontogenically distinct cell subtypes and their associated lineages. The application of Cre-based genetic targeting to microglia biology has had particularly important impacts on our understanding of brain immunity. Over the past decade, research utilizing two Cx3Cr1 CreER lines 16,35 helped to elucidate the ontogeny of microglia and non-microglia CNSassociated macrophages, and the roles of specific microglia genes and signaling pathways in brain development and function 35,36 . Recently, in an effort to improve the specificity of brain microglia gene manipulation, several new brain microglia-specific inducible CreER mouse lines were generated, including Tmem119 CreER , P2ry12 CreER Sall1 CreER , HexB CreER 8,10,31,37 . Since the initial reports characterizing these new lines, we and others noticed different degrees of leakiness and recombination efficiency. Furthermore, recent reports document important off-target effects in Cx3cr1 CreER(Litt) mice 12,27 , prompting us to perform a more detailed characterization of the publiclyavailable microglia-targeting CreER lines. Our studies focus on five key characteristics of CreER gene targeting: specificity, leakiness, efficiency, extra-neural recombination, and off target effects.
Because Sall1 CreER and Hexb CreER were not readily available to us at the time of this report, we compare our results to published studies using these lines 31,37 . We believe this evaluation will provide valuable information to the field, particularly for researchers aiming to identify appropriate mouse models for conditional gene deletion in microglia.
. CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted April 17, 2023. ; https://doi.org/10.1101/2023.04.17.536878 doi: bioRxiv preprint in the brain, and can distinguish CNS associated macrophages (CAMs) from microglia. Sall1 CreER recombines microglia specifically, in addition to neurons, astrocytes, and oligodendrocytes.
Leakiness: We found that Cx3cr1 CreER lines recombined the more sensitive Ai9 tdTomato reporter a relatively large number of microglia (and splenic macrophages) in the absence of tamoxifen, whereas Tmem119 CreER and P2ry12 CreER lines were comparatively less leaky. Sall1 CreER is relatively more leaky than Hexb CreER , and similar to Cx3cr1 CreER(Jung) . These three lines were directly compared using both RosaYFP and Ai9 reporter mice to assess leak 37 . Like us, this group observed that leakiness was negatively correlated with recombination efficiency or the length of the loxP flanked region (higher leakiness in Ai9 reporter and lower leakiness in R26-YFP reporter).
Efficiency: Efficiency was directly correlated with inter-loxP distance and intrinsic CreER activity/expression. Tmem119 CreER and P2ry12 CreER mice with the Ai14 reporter (0.9kb) maintained high recombination efficiency but only achieved partial recombination in the R26-YFP reporter (2.7kb) allele (about 30% of all microglia population), reiterating the importance of loxP distance in recombination efficiency. In line with this conclusion, we found that the Cx3Cr1 CreER(Jung) line effectively deletes both copies of the shorter Tgfb1 fl/fl target gene and the longer Alk5 fl/fl gene while the P2ry12 CreER had a 50% decrease in mRNA levels of these two genes. It is worth noting that this 50% gene deletion efficiency in the P2ry12 CreER line was evaluated in sorted YFP+ microglia . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted April 17, 2023. ; https://doi.org/10.1101/2023.04.17.536878 doi: bioRxiv preprint populations. As P2ry12 CreER only achieves R26-YFP recombination in about 30% of total microglia, the gene deletion efficiency in the full population of microglia sorted using the Ai9 tdTomato reporter was lower (20% decrease in Alk5 mRNA levels in total tdT + cells). Masuda et.
al 37 directly compared HexB CreER , Sall1 CreER and Cx3cr1 CreER(Jung) , and found HexB CreER and Sall1 CreER to be similarly less efficient than Cx3cr1 CreER . Note that most of their analysis was done using homozygous Hexb CreER/CreER mice which likely increased recombination efficiency, as we observed with P2ry12 CreER/CreER mice. was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted April 17, 2023. ; https://doi.org/10.1101/2023.04.17.536878 doi: bioRxiv preprint that despite robust activation and loss of P2RY12 expression in microglia in Cx3cr1 CreER(Litt) mice, pSMAD3 immunofluorescence intensity (a measure of TGF-β signaling) was not appreciably changed. Furthermore, we observed no evidence of alterations in microglia homeostatic gene expression in Cx3cr1 CreER(Jung) and P2ry12 CreER lines.
We and others previously investigated whether the gene knock-in strategies used to generate these various CreER lines might affect gene expression resulting in unintended effects. AitdTomato or R26-YFP reporter lines can be easily accomplished to facilitate genetic reporterbased FACS sorting. As peripheral macrophages also express Cx3Cr1 and are therefore recombined by Cx3cr1 CreER , peripheral immune effects are a potential confound that needs to be considered when using these two lines.
To investigate gene function in "true" microglia, Tmem119 CreER , P2ry12 CreER are preferable. Additional consideration should be taken with Hexb CreER which was found to strongly recombine blood cells. Utilization of the Tmem119 CreER or P2ry12 CreER lines therefore enables enriched labeling of CNS parenchyma microglia and allows researchers to track endogenous microglia and study their specific properties after CNS injury or in neurodegenerative conditions. This was successfully achieved in an EAE model using the P2ry12 CreER line 19 . Future use of these tools could facilitate the pre-labeling of brain microglia cells and allow for the separation of microglia from infiltrating peripheral monocytes/macrophage by FACS sorting. This will help resolve a long-standing question in the field: whether the infiltrating monocytes/macrophage have a distinct neuroinflammatory program compared to the resident brain microglia in CNS disease or injury. The lower recombination efficiency observed in P2ry12 CreER and Tmem119 CreER also lends itself well to performing mosaic genetic experiments which allow for in vivo single-cell phenotypic analyses, where differences among single cells can be attributed to induced gene mutation or expression in an otherwise identical organism and genetic background.
We attempted to enhance recombination efficiency of the P2ry12 CreER line by crossing to the iSuReCre reporter mouse. Compared to DCX CreER ;iSuReCre mice, with robust recombination of immature DCX+ neuroblasts, we observed no microglial recombination in P2ry12CreER;iSuReCre mice based on immunostaining brain sections for the iSuReCre membranous tomato report. Interestingly, iSuReCre shows reliable recombination and tdTomato expression in peripheral macrophages by LysMCre, and Tie2Cre;iSuReCre mice have apparently strong recombination of retinal microglia. The underlying cause for lack of iSuReCre recombination by P2ry12 CreER mice is unknown but may be due to the genomic integration site of . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted April 17, 2023. ; https://doi.org/10.1101/2023.04.17.536878 doi: bioRxiv preprint the iSuReCre-tdT construct which has not been as well-characterized as the ROSA26 safe-harbor locus. Our data in the Cx3cr1 CreER(Jung) -iSureCre mice show that even with the strong Cx3cr CreER driver, MbTomato expression is absent in microglia (Data not shown), therefore supporting the hypothesis that the specific loci where iSuRe-Cre cassette is inserted might be silenced in microglia. An alternative method to boost CreER activity is to breed the line to homozygosity.
P2ry12 CreER/CreER (unpublished observations) and Hexb CreER/CreER mice have more efficient gene deletion. However, this may come at the cost of increased leakiness and potential off-target effects related to gene dosage. Additionally, an investigator can choose to breed in one knock-out allele to reduce the number of recombination events required for complete gene deletion.
When using the Tmem119 CreER or the P2ry12 CreER lines, we recommend using a reporter with inter-loxP distance as close to that of the target gene as possible, to include one knockout allele so that only one recombination event is required for gene deletion (if heterozygous knockout mice do not cause phenotype), or ideally to utilize one target allele that is floxed with an intrinsic reporter (knock-in/knock-out construct) and one knockout allele to simultaneously facilitate gene deletion and precisely identify knockout cells. Understanding that null and conditional knockoutknockout reporters, and other Cre "boosting" constructs, are not readily available, additional analysis such as qRT-PCR on sorted microglia, RNAscope or immunohistochemical staining is recommended to validate successful knockout or knockdown of the target genes in brain microglia.
We recommend performing this validation in a small pilot study before investing time and resources in larger scale experiments.
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. Together with this study, we hope our comprehensive comparison and analysis provides important data and guidance for researchers interested in performing conditional gene manipulation in microglia.
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Declaration of interests
The authors declare no competing interests.
Supplementary material is available online. Table S1. Quantification of the FACS analysis of the reporter+ microglia in whole brain single cell suspension prepared from the P2RY12CreER-Ai9-R26-YFP mice after TAM treatment.  was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted April 17, 2023. ; https://doi.org/10.1101/2023.04.17.536878 doi: bioRxiv preprint of 1 animal (the average for each animal is obtained by quantifying multiple brain sections at similar anatomical location) and the average for each animal was used as a single data point for statistical analysis. **p < 0.01 and ***p < 0.001, for Two way ANOVA analysis, Tukey post-hoc pair wise analysis. Ai9 vs R26-YFP is significantly different as a factor (p<0.001). Data were combined from 2 independent cohorts of mice. Scale bar: 100 μm. Compared to the two Cx3cr1 CreER lines (Littman and Jung), TMEM119 CreER and P2ry12 CreER show less leakiness in the absence of TAM but a decreased recombination efficiency and mosaic recombination in microglia.    Each data point represents the average of 1 animal (the average for each animal is obtained by quantifying multiples pleen sections) and the average for each animal was used as a single data point for statistical analysis. **p < 0.01 and ***p < 0.001, for Two-way ANOVA analysis, Tukey post-hoc pairwise analysis. Ai9 vs R26-YFP is significantly different as a factor (p<0.001 for TAM treated group). Data were combined from 2-3 independent cohorts of mice. Scale bar: 100 μm.  Students T-Test). Data were combined from 3 vehicle-treated and 4 tamoxifen-treated mice. Scale bar: 100 μm.

Lead contact
Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact, Yu Luo (luoy2@ucmail.uc.edu).

Materials availability
This study did not generate new unique reagents.

Data and code availability
• We analyzed recently published publicly available RNA-seq data sets: GEO: GSE190207 and GEO: GSE124868 • Microscopy data and behavioral test data reported in this paper will be shared by the lead contact upon request.
• No original code was generated in this study.
• Any additional information required to reanalyze the data reported in this paper is available from the lead contact upon request.
. CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted April 17, 2023. No animals were excluded from data analysis except where tissue preservation quality was not ideal due to unsuccessful perfusion (indicated by shredded tissue) or poor cryoprotection in tissues.

Tamoxifen treatment in vivo.
The variety of MG CreER -Ai9 or MG CreER -R26YFP or P2ry12 CreER -Ai9/R26YFP double reporter mice and the TGF-β1 and ALK5 floxed mice (8-12 weeks old, both male and females) were given tamoxifen (TAM) dissolved in 10% EtOH/90% sunflower oil or vehicle without TAM by gavage feeding at a dose of 180 mg/kg daily for 5 consecutive days. This dosing regimen was previously demonstrated to provide maximal recombination with minimal mortality and successfully monitored the adult NSCs in previous studies by our group and the others 47,48 . To evaluate recombination efficiency in the brain and the clearance of splenic monocytes/macrophages, we . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted April 17, 2023. ; https://doi.org/10.1101/2023.04.17.536878 doi: bioRxiv preprint collected the brain and the spleen of VEH or TAM treated mice at 4 weeks after the treatment for immunohistochemical (IHC) or flow cytometry (FACS) analysis. For studying the phenotype of non-homeostatic microglia in CreER(+/WT) mice with TAM treatment, Cx3cr1-CreER(Litt)(+/WT) neonatal mice were subjected to either VEH or TAM treatment at neonatal days of P1-P4 (50µg via intragastric injection) and brain harvested for analysis at p15. To test effect of TAM treatment in the Cx3cr1-CreER(Litt)(+/WT) adolescent mice, mice at 3 weeks of age were treated with 5 day gavage (180mg/kg daily) and harvested at 4 weeks after TAM treatment. Neonatal Cx3cr1 CreER(Jung) allele carrying mice (Cx3cr1 CreER(Litt)(+/WT) tgfbr2 wt/fl or tgfb1 WT/fl ) or P2ry12 CreER allele carrying mice (P2ry12 CreER+/WT tgfb1 WT/fl ) were subjected to TAM treatment at neonatal days of P1-P4 (50µg via intragastric injection) and brain harvested for analysis at P15 or at neonatal days P4-P6 (500ug via intraperitoneal injection) and harvested at P30.

Immunohistochemistry
Mice were anesthetized and perfused with PBS or PBS followed by 4% paraformaldehyde (PFA).
For brains that were subjected to both IHC and FACS analysis, mice were only perfused with PBS and part of the brain block was drop fixed in 4% PFA overnight before being transferred to 20% then 30% sucrose. For mice that were perfused by 4% PFA, the brain and spleen was dissected and post-fixed in 4% PFA overnight at 4 °C and equilibrated in 20% then 30% sucrose. 30 μmthick sections were cut in a Leica Cryostat and blocked in 4% BSA/0.3% Triton-x100 for 1 hour.
After blocking, sections were incubated with primary antibodies for 18h-42h at 4 °C and followed by appropriate secondary antibodies conjugated with Alexa fluorescence 488, 555, 647 or 790.
The following primary antibodies were used in this study: IBA1 (Rabbit 1:1000 Wako), P2RY12 was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted April 17, 2023. ; images were taken at 20X or 40X objectives. Quantification of the percentage of reporter positive cells (tdTomato+ or YFP+) among IBA1+ microglia (in the brain) or macrophage (in the spleen) was carried out by as described in our previous publication 49 Figure 1. Evaluation of the TAM-independent leakiness and the efficiency of TAM-dependent cre recombination in the four different creER driver lines using either the Ai9 (tdTomato) or R26-YFP reporter mouse lines. The experimental timeline is shown in panel (A). Representative images from each cre driver and reporter line (B-V for VEH treatment and b-v for TAM treatment). Cre driver and the reporter line are indicated on the left side of the panels. Quantification of reporter+ cells in the IBA1+ populations in the brain is shown in panel W (for VEH treatment) and w (for TAM treatment). Representative images are taken from the cortical region which reflects the general and homogenous trend in the whole parenchyma. Each data point represents the average of 1 animal (the average for each animal is obtained by quantifying multiple brain sections at similar anatomical location) and the average for each animal was used as a single data point for statistical analysis. **p < 0.01 and ***p < 0.001, for Two way ANOVA analysis, Tukey post-hoc pair wise analysis. Ai9 vs R26-YFP is significantly different as a factor (p<0.001). Data were combined from 2 independent cohorts of mice. Scale bar: 100 μm. Compared to the two Cx3cr1 CreER lines (Littman and Jung), TMEM119 CreER and P2ry12 CreER show less leakiness in the absence of TAM but a decreased recombination efficiency and mosaic recombination in microglia.
. CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted April 17, 2023.  In contrast, in the DCXC reER -iSuReCre mice that are treated with TAM, at 5 days post TAM treatment, DCX+ immature neuroblasts are labeled with MbTomato protein (white arrows) and at 30 days post TAM treatment, MbTomato expression are mostly detected in DCX-NeuN+ mature neurons (yellow arrows), supporting that the iSuRe-Cre construct is able to be induced in a cohort of immature neuroblasts which mature later into NeuN+ neurons in the dentate gyrus of adult mice. Scale bar=100 µm.
. CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted April 17, 2023. ; https://doi.org/10.1101/2023.04.17.536878 doi: bioRxiv preprint . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted April 17, 2023. ; https://doi.org/10.1101/2023.04.17.536878 doi: bioRxiv preprint Figure 5. Evaluation of the splenic TAM-independent and TAM-dependent cre recombination in the four different creER driver lines using either the Ai9 (tdTomato) or R26-YFP reporter mouse lines. The experimental timeline is shown in panel (A). Representative images from each cre driver and reporter line (B-V for VEH treatment and b-v for TAM treatment). Cre driver and the reporter line are indicated on the left side of the panels. Quantification of reporter+ cells in the IBA1+ populations in the spleen is shown in panel W (for VEH treatment) and w (for TAM treatment). Each data point represents the average of 1 animal (the average for each animal is obtained by quantifying multiples pleen sections) and the average for each animal was used as a single data point for statistical analysis. **p < 0.01 and ***p < 0.001, for Two-way ANOVA analysis, Tukey post-hoc pairwise analysis. Ai9 vs R26-YFP is significantly different as a factor (p<0.001 for TAM treated group). Data were combined from 2-3 independent cohorts of mice. Scale bar: 100 μm.
. CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted April 17, 2023. ; https://doi.org/10.1101/2023.04.17.536878 doi: bioRxiv preprint Figure 6. Evaluation of the dyshomeostatic microglia in different microglia-specific CreER drivers after TAM treatment at different ages. P2Ry12 expression is used as a measure of dyshomeostasis in microglia. (A), consistent with previous studies, we observe dyshomeostasis of microglia (indicated by loss of P2RY12 expression) across many regions in the neonatal Cx3cr1 CreER(Litt) (+/WT) mice treated with TAM. This phenotype is not observed in (B) the adolescent (3wk old) Cx3cr1 CreER(Litt) (+/WT) mice that received TAM treatment or (C) neonatal Cx3cr1 CreER(Jung) (+/WT) and P2RY12CreER (+/WT) mice that received TAM at the similar time frame. Scale bar= 100µm.
. CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted April 17, 2023. ; . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted April 17, 2023. ; https://doi.org/10.1101/2023.04.17.536878 doi: bioRxiv preprint Supplementary Figure 2. FACS analysis of reporter-positive cells in total brain single cell resuspension confirms the reporter recombination efficiency differences among the three investigated CreER lines. tdTomato+ cell percentage from all three different lines show similar high-efficiency recombination and the YFP+ cells show higher recombination efficiency in the CX3CR1CreERJung-R26-YFP mice but significantly lower recombination efficiency in the P2RY12 CreER -R26-YFP mice and TMEM119 CreER -R26-YFP line. Each data point represents data from one animal. **p < 0.01 and ***p < 0.001, for Two way ANOVA analysis, Tukey post-hoc pairwise analysis. Data were combined from 2-3 independent cohorts of mice for each line. . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted April 17, 2023. ; https://doi.org/10.1101/2023.04.17.536878 doi: bioRxiv preprint