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

Abstract

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 (Cx3cr1^CreER(Litt), Cx3cr1^CreER(Jung), P2ry12^CreER, Tmem119^CreER), 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.

Keywords: Cre recombinase; Cx3cr1; Microglia; P2ry12; TMEM119; inducible gene recombination.

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.

Figure 2.

Independent recombination of the two floxed alleles on a single cell level in the P2RY12^CreER double reporter (Ai9:R26-YFP) mouse line. Evaluation of the TAM-dependent recombination of either Ai9-tdTomato or R26-YFP allele which are both located in the ROSA26 loci in a double reporter mouse in the P2RY12CreER line suggests that although on a populational level, Ai9-tdTomato reporter has a higher probability of being recombined than the R26-YFP allele, on a single cell level, the recombination of each individual allele can be independent and does not always follow the size of the floxed region rule. (A), experimental timeline. (B-E), IHC evaluation of the recombination of microglia on either of the reporter expression. Note tdTomato+:YFP+ double positive microglia (orange arrow), more abundant tdTomato+:YFP− microglia (white arrow) and the less abundant YFP+:tdTomato- microglia (white arrowhead). (F-G), representative FACS plots for no color control or the double reporter flow analysis. For quantification of % of each population, see Supplementary Table 1.

Figure 3.

Evaluation of the gene deletion efficiency on distinct homozygous floxed target gene alleles in the Cx3cr1CreER(Jung) and P2Ry12CreER drivers using quantitative RT-PCR. (A-D). Animal genotype and experimental flow. Total mRNA levels are evaluated for the floxed exon in the TGFb1 gene in YFP+ microglia sorted from the Cx3cr1^CreER(Jung) or P2RY12^CreER—TGFb1 ^fl/fl-R26-YFP mice at 3 weeks after TAM treatment. (E-G). Total mRNA levels are evaluated for the floxed exon in the ALK5 gene in either YFP+ microglia sorted from the Cx3cr1^CreER(Jung) or P2RY12^CreER—ALK5 ^fl/fl-R26-YFP mice or tdTomato+ microglia from the P2RY12^CreER—ALK5 ^fl/fl-Ai9 mice at 3 weeks after TAM treatment. Each data point represents the average of 1 animal (the average for each animal is obtained by averaging 3 technical replication of the qRT-PCR reaction for that animal) and the average for each animal was used as a single data point for statistical analysis. **p < 0.01 and ***p < 0.001, **** p<0.0001 for Two way ANOVA analysis, Tukey post-hoc pairwise analysis.

Figure 4.

iSuRe-Cre mouse line successfully induces constitutive cre-P2A-MbTomato expression in DCX^CreER mouse line but not in the P2ry12^CreER mouse line after TAM treatment. (A) Illustration of mouse transgene constructs and experimental timeline. (B-E) in the absence of TAM, there is no MbTomato expression in microglia with ectopic MbTomato expression in cells that demonstrates typical neuron morphology in cortex and striatum. (F-G), treatment of TAM in mice does not induce MbTomato expression in microglia and presents with similar neuronal ectopic expression of MbTomato. (J-R). 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.

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.

Figure 6.

Evaluation of the dyshomeostatic microglia in different icroglia-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 region 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.

Figure 7.

Similarity in microglia phenotypes from neonatal tamoxifen-induced Cx3cr1CreER(Litt) and Tgfbr2fl/fl;Cx3cr1Cre mice. (A) Transcriptomic phenotypes from P15 Cx3cr1CreER(Litt) (vehicle versus tamoxifen) and Tgfbr2;Cx3cr1Cre (Tgfbr2fl/+;Cre vs Tgfbr2fl/fl;Cre) mice showing changes in INF-signaling related, MgND/DAM, and hemeostatic gene expression. (B) Correlation between two data sets. (C) Brain sections from P15 Cx3cr1CreER(Litt) (vehicle versus tamoxifen) mice stained for DAPI (blue), IBA1 (green), P2ry12 (grey), pSMAD3 (red). Quantification of pSMAD3 mean fluorescence intensity in individual microglia from each group (D) shows no significant difference in pSMAD3 (P=0.0644; Students T-Test). Data were combined from 3 vehicle-treated and 4 tamoxifen-treated mice. Scale bar: 100 μm.