Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2023 Sep 26;42(9):113031.
doi: 10.1016/j.celrep.2023.113031. Epub 2023 Aug 26.

A comparative analysis of microglial inducible Cre lines

Travis E Faust  1 , Philip A Feinberg  1 , Ciara O'Connor  1 , Riki Kawaguchi  2 , Andrew Chan  3 , Hayley Strasburger  3 , Maximilian Frosch  4 , Margaret A Boyle  1 , Takahiro Masuda  5 , Lukas Amann  4 , Klaus-Peter Knobeloch  6 , Marco Prinz  7 , Anne Schaefer  8 , Dorothy P Schafer  9
Affiliations

A comparative analysis of microglial inducible Cre lines

Travis E Faust et al. Cell Rep. .

Abstract

Cre/loxP technology has revolutionized genetic studies and allowed for spatial and temporal control of gene expression in specific cell types. Microglial biology has particularly benefited because microglia historically have been difficult to transduce with virus or electroporation methods for gene delivery. Here, we investigate five of the most widely available microglial inducible Cre lines. We demonstrate varying degrees of recombination efficiency, cell-type specificity, and spontaneous recombination, depending on the Cre line and inter-loxP distance. We also establish best practice guidelines and protocols to measure recombination efficiency, particularly in microglia. There is increasing evidence that microglia are key regulators of neural circuits and major drivers of a broad range of neurological diseases. Reliable manipulation of their function in vivo is of utmost importance. Identifying caveats and benefits of all tools and implementing the most rigorous protocols are crucial to the growth of the field and the development of microglia-based therapeutics.

Keywords: CP: Neuroscience; Cre/loxP; CreER; microglia; recombination.

PubMed Disclaimer

Conflict of interest statement

Declaration of interests The authors declare no competing interests.

Figures

Figure 1.
Figure 1.. Recombination efficiency varies across microglial CreER lines
(A) Diagram of transgenes used to express CreER in Cx3cr1YFP–CreER (Litt), Cx3cr1CreER (Jung), Tmem119CreER, HexbCreER, and P2ry12CreER mice. (B) Diagram of the Rosa26mTmG allele and corresponding cellular fluorescence before and after Cre/loxP DNA recombination. loxP sites are indicated by yellow triangles. (C) Diagram of experimental protocol used to assess tamoxifen (TAM)-induced Cre/loxP recombination of Rosa26mTmG/+ in microglia by flow cytometry. (D–H) Representative flow cytometry results show the percentage of mGFP+ (mG+) and mTomato+ (mT+) microglia from individual animals from each group. (I) Quantification of the percentage of recombined mGFP+ microglia in microglial CreER lines showed increased recombination in TAM vs. oil for all five CreER lines (Student’s t tests: Cx3cr1YFP–CreER/+ (Litt):n = 5 oil, 5 TAM mice; Cx3cr1CreER/+ (Jung):n = 5 oil, 4 TAM mice; Tmem119CreER/+:n = 8 oil, 7 TAM mice; HexbCreER/+: n = 9 oil, 10 TAM mice; P2ry12CreER/+: n = 4 oil, 4 TAM mice; ****p < 0.0001). All data are presented as mean ± SEM. Individual data points indicate males (squares) and females (circles). See also Figures S1 and S2 and Table S1.
Figure 2.
Figure 2.. Spatial distribution of Rosa26mTmG recombination in microglial CreER lines
(A, C, E, G, and I) Representative immunofluorescent images of brain sections from right hemispheres of oil- and TAM-injected mice used for flow cytometry analysis in Figure 1. Sections were immunolabeled for anti-P2RY12 (Alexa Fluor 647 [AF647] pseudo-colored red) to identify microglia and anti-GFP (green) to identify recombined cells. Scale bars, 50 μm. (B, D, F, H, and J) Quantifications of the percentage of P2RY12+ microglia that are mGFP+ in the cortex, hippocampus, thalamus, cerebellum, and brainstem of each CreER line after exposure to oil or TAM (B: Cx3cr1YFP–CreER (Litt), n = 3, 3 mice; D: Cx3cr1CreER (Jung), n = 4, 3 mice; H: HexbCreER, n = 4, 4 mice; J: P2ry12CreER, n = 4, 4 mice; two-way repeated-measures ANOVA with Tukey’s post hoc test, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001). (K–O) Quantifications of the number of P2RY12− cells per section that are mGFP+ in the cortex, hippocampus, thalamus, cerebellum, and brainstem of each CreER line after exposure to oil or TAM (K: Cx3cr1YFP–CreER (Litt), n = 3, 3 mice; L: Cx3cr1CreER (Jung), n = 4, 3 mice; M: Tmem119CreER, n = 4, 3 mice; N: HexbCreER: n = 4, 4 mice; O: P2ry12CreER: n = 4, 4 mice; two-way repeated measures ANOVA with Tukey’s post hoc test). All data are presented as mean ± SEM. Individual data points indicate males (squares) and females (circles). See also Figure S3.
Figure 3.
Figure 3.. Cell-type specificity of microglial CreER lines
(A) Representative immunofluorescent images of brain sections from right hemispheres of oil- and TAM-injected Rosa26mTmG/+;Cre-DriverCreER/+ mice used for flow cytometry analysis in Figure 1. Sections were immunolabeled for anti-LYVE1 (AF647 pseudo-colored red) to identify border-associated macrophages and anti-GFP (green) to identify recombined cells. Arrows indicate mGFP+ border-associated macrophages. Asterisks indicate mGFP+ cells along the meninges immunonegative for anti-LYVE1. Scale bars, 50 μm. (B) Quantification of the percentage of recombined mGFP+ border-associated macrophages shows increased recombination in TAM-injected Cx3cr1CreER lines compared with Tmem119CreER, HexbCreER, and P2ry12CreER lines (two-way ANOVA with Tukey’s post hoc test; n = 3 oil Cx3cr1YFP–CreER (Litt), 3 TAM Cx3cr1YFP–CreER (Litt), 4 oil Cx3cr1CreER (Jung), 3 TAM Cx3cr1CreER (Jung), 4 oil Tmem119CreER, 3 TAM Tmem119CreER, 4 oil HexbCreER, 4 TAM HexbCreER, 4 oil P2yr12CreER, and 4 TAM P2ry12CreER mice; ***p < 0.001, ****p < 0.0001). (C) Quantification of the frequency of recombined mGFP+ cells immunonegative for anti-LYVE1 along the meningeal border shows increased recombination in Tmem119CreER mice (two-way ANOVA with Tukey’s post hoc test; n = 3 oil Cx3cr1YFP–CreER (Litt), 3 TAM Cx3cr1YFP–CreER (Litt), 4 oil Cx3cr1CreER (Jung), 3 TAM Cx3cr1CreER (Jung), 4 oil Tmem119CreER, 3 TAM Tmem119CreER, 4 oil HexbCreER, 4 TAM HexbCreER, 4 oil P2yr12CreER, and 4 TAM P2ry12CreER mice; ****p < 0.0001). (D and E) Representative immunofluorescent images of brain sections from right hemispheres of oil- and TAM-injected Rosa26mTmG;Tmem119CreER mice used for flow cytometry analysis. Sections were immunolabeled for anti-GFP (green) to identify recombined cells and (D) anti-ALDH1A2 (AF647 pseudo-colored red) to identify arachnoid meningeal fibroblasts or (E) anti-S100A6 (AF647 pseudo-colored red) to identify pial meningeal fibroblasts. Arrows indicate mGFP+ cells along the meninges. Scale bars, 50 μm. All data are presented as mean ± SEM. Individual data points indicate males (squares) and females (circles).
Figure 4.
Figure 4.. Off-target effects of TAM in Cx3cr1YFP–CreER (Litt) mice
(A) Timeline of neonatal TAM injection for images in (B) and (C). (B) Fluorescent images of brain sections from Cx3cr1YFP–CreER/+ (Litt), Tmem119CreER/+, and Cx3cr1EGFP/+ mice injected with TAM neonatally, immunolabeled with anti-P2RY12. Large regions of the cortex are devoid of anti-P2RY12 immunofluorescence in Cx3cr1YFP–CreER/+ (Litt) mice, but not Tmem119CreER/+ or Cx3cr1EGFP/+ mice. Scale bars, 500 mm; 200 μm (insets). (C) Fluorescent images of brain sections from Cx3cr1YFP–CreER/+ (Litt) mice injected with TAM neonatally, immunolabeled with anti-P2RY12 (red) and anti-GFP to identify Cx3cr1YFP+ cells (green). Regions of the cortex devoid of anti-P2RY12 immunofluorescence (dotted outline in inset) contain Cx3cr1YFP+ cells with a morphology characteristic of reactive microglia. Scale bars, 500 μm; 100 μm (insets). (D) Diagram of experimental protocol used to assess the effect of TAM injection at P28 on anti-P2RY12 immunofluorescence in Cx3cr1YFP–CreER/+ (Litt);Rosa26mTmG/+ mice. (E and F) Fluorescent images of brain sections from Cx3cr1YFP–CreER/+ (Litt);Rosa26mTmG/+ mice used for flow cytometry analysis in Figure 1, immunolabeled with anti-P2RY12 (imaged using AF647). No patches are devoid of anti-P2RY12 immunofluorescence. Scale bars, 500 μm (E); 200 μm (F). (G) Quantification of the percentage of mice with patches devoid of anti-P2RY12 immunofluorescence shows higher prevalence in Cx3cr1YFP–CreER/+ (Litt) mice injected with TAM as neonates vs. Cx3cr1YFP–CreER/+ (Litt) mice injected with TAM at P28 (chi-squared test: n = 4, 4 mice; **p < 0.01). (H) Diagram of experimental protocol used to perform RNA sequencing on Cx3cr1YFP–CreER/+ (Litt) mice injected with TAM or oil at P28. (I) Smear plot of TAM-vs. oil-injected Cx3cr1YFP–CreER/+ (Litt) mice depicting log fold change (FC) on the y axis against log counts per million (CPM) on the x axis (n = 4, 5 mice). Differentially expressed genes with false discovery rate (FDR) < 0.05 are annotated in red (upregulated by TAM) or blue (downregulated by TAM). See also Figure S4.
Figure 5.
Figure 5.. Spontaneous recombination of Rosa26mTmG in microglial CreER lines
(A) Diagram of experimental protocol used to assess spontaneous Cre/loxP recombination of Rosa26mTmG/+ in microglia by flow cytometry and immunofluorescence. (B) Representative flow cytometry results show the percentage of recombined mGFP+ (mG) and mTomato+ (mT) microglia from individual animals from each group from Figure 1 and uninjected (no oil) Cx3cr1YFP–CreER/+ (Litt) mice. (C) Quantification of the percentage of recombined mGFP+ microglia shows increased spontaneous recombination of the Rosa26mTmG allele in the Cx3cr1YFP–CreER (Litt) line compared with the Cx3cr1CreER (Jung), Tmem119CreER, HexbCreER, and P2ry12CreER lines (one-way ANOVA with Tukey’s post hoc test; n = 5 Cx3cr1YFP–CreER/+ (Litt), 5 Cx3cr1CreER/+ (Jung), 8 Tmem119CreER/+, 9 HexbCreER/+, and 4 P2ry12CreER/+ mice; *p < 0.05, ****p < 0.0001). (D) Representative immunofluorescent images of brain sections from right hemispheres of oil-injected mice used for flow cytometry analysis in (B) and (C). Sections were immunolabeled for anti-P2RY12 (AF647 pseudo-colored red) to identify microglia and anti-GFP (green) to identify recombined cells. The number of recombined mGFP+ microglia (white arrows) matches the results observed by flow cytometry. In the Cx3cr1YFP–CreER/+ (Litt) line, the soma of non-recombined microglia are also immunolabeled by anti-GFP because of the constitutive expression of YFP (asterisks), but it can be distinguished from recombined mGFP+ microglia by fluorescence intensity and membrane labeling. Scale bars, 50 μm. All data are presented as mean ± SEM. Individual data points indicate males (squares) and females (circles). See also Figure S5.
Figure 6.
Figure 6.. Inter-loxP distance is a determinant of recombination efficiency in microglial CreER lines
(A) Diagram of the Rosa26Ai allele and Rosa26mTmG allele before and after Cre/loxP recombination shows the locations of the loxP sites (yellow triangles) and the inter-loxP distance. (B) Diagram of experimental protocol used to assess TAM-induced Cre/loxP recombination of Rosa26Ai and Rosa26mTmG in microglia by flow cytometry. (C, D, F, and G) Representative flow cytometry results from individual oil- and TAM-injected Cx3cr1YFP–CreER/+ (Litt) and Tmem119CreER/+ mice expressing Rosa26Ai9/+ are compared with flow cytometry results of Rosa26mTmG/+ from Figure 1. Recombined Rosa26Ai microglia are tdTomato+, whereas recombined Rosa26mTmG microglia are mGFP+ (mG) and non-recombined Rosa26mTmG microglia are mTomato+ (mT). (E and H) Quantifications of recombination of Rosa26Ai and Rosa26mTmG in microglia from oil- and TAM-injected mice show significantly more recombination of the 0.9 kb Rosa26Ai floxed allele vs. the 2.4 kb Rosa26mTmG floxed allele in (E) Cx3cr1YFP–CreER/+ (Litt) mice (two-way ANOVA with Sidak’s post hoc test; n = 4 Rosa26Ai oil, 5 Rosa26mTmG oil, 4 Rosa26Ai TAM, and 5 Rosa26mTmG mice; ****p < 0.0001) and (H) Tmem119CreER/+ mice (two-way ANOVA with Sidak’s post hoc test; n = 4 Rosa26Ai oil, 8 Rosa26mTmG oil, 4 Rosa26Ai TAM, and 7 Rosa26mTmG mice; ****p < 0.0001). (I) Diagram of protocol to assess Cre/loxP recombination in microglia by endpoint PCR of genomic DNA (gDNA). (J and K) Gel images of endpoint PCR products from microglial gDNA isolated by florescence-activated cell sorting (FACS) from oil- and TAM-injected Cx3cr1YFP–CreER/+ (Litt) and Tmem119CreER/+ mice expressing Rosa26Ai9/+ or Rosa26mTmG/+. All data are presented as mean ± SEM. Individual data points indicate males (squares) and females (circles). See also Figure S6.
Figure 7.
Figure 7.. A quantitative PCR (qPCR) protocol to quantitatively assess recombination in microglia
(A) Diagram of protocol used to quantify Cre/loxP recombination of microglial gDNA by qPCR. (B) Diagram of experiment to assess Cre/loxP recombination in mice injected with TAM or oil. (C) Quantification of the percentage of recombined gDNA by qPCR shows increased recombination in TAM vs. oil for all three CreER lines (Rosa26mTmG/+;Cx3cr1YFP–CreER/+ (Litt): Student’s t test, n = 4 oil, 4 TAM mice; Cx3cr1CreER/+ (Jung): Student’s t test, n = 4 oil, 4 TAM mice; Tmem119CreER/+: Student’s t test, n = 4,oil, 3 TAM mice; *p < 0.05, ***p < 0.001, ****p < 0.0001). (D) Graph of percent recombination of Rosa26mTmG in microglia in mice injected with TAM or oil as measured by flow cytometry analysis (see also Figure 1) vs. the recombination rate as measured by qPCR of microglial gDNA isolated by FACS. Data points fit to a linear curve (black line; r = 0.9267), closely aligned with the line of identity (red dashed line), indicating that qPCR provides a linear, quantitative measurement of Rosa26mTmG recombination in in vivo samples. Data in (C) are presented as mean ± SEM. Individual data points in (C) indicate males (squares) and females (circles). See also Figure S7.
See this image and copyright information in PMC

Update of

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Wu Y, Dissing-Olesen L, MacVicar BA, and Stevens B (2015). Microglia: Dynamic Mediators of Synapse Development and Plasticity. Trends Immunol. 36, 605–613. 10.1016/j.it.2015.08.008. - DOI - PMC - PubMed
    1. Prinz M, Jung S, and Priller J (2019). Microglia Biology: One Century of Evolving Concepts. Cell 179, 292–311. 10.1016/j.cell.2019.08.053. - DOI - PubMed
    1. Salter MW, and Stevens B (2017). Microglia emerge as central players in brain disease. Nat. Med. 23, 1018–1027. 10.1038/nm.4397. - DOI - PubMed
    1. Biber K, Möller T, Boddeke E, and Prinz M (2016). Central nervous system myeloid cells as drug targets: current status and translational challenges. Nat. Rev. Drug Discov. 15, 110–124. 10.1038/nrd.2015.14. - DOI - PubMed
    1. Hansen DV, Hanson JE, and Sheng M (2018). Microglia in Alzheimer’s disease. J. Cell Biol. 217, 459–472. 10.1083/jcb.201709069. - DOI - PMC - PubMed

Publication types

LinkOut - more resources