Skip to main content
. 2021 May 12;41(19):4349–4365. doi: 10.1523/JNEUROSCI.1940-20.2021

Figure 3.

Figure 3.

TLR4 cKO is tamoxifen-dependent and microglia-specific. A, Cx3CR1-CreERT2-eYFP; tdTomatoLSL mice were generated in which all Cx3CR1+ cells express the tdTomato transgene after tamoxifen-dependent Cre recombination. Differential turnover rates of central microglia versus peripheral macrophages result in persistent tdTomato expression in microglia. B, Flow cytometry analysis of CNS microglia (gated live cells: CD19– CD3– CD45mid CD11bhi Cx3CR1-YFP+) from Cx3CR1-CreERT2-eYFP; R26-tdTomatoLSL mice demonstrates tamoxifen-dependent and persistent expression of the tdTomato transgene. C, Flow cytometry analysis of spleen macrophages (gated live cells: CD19– CD3– CD45hi CD11bhi Cx3CR1-YFP+) from Cx3CR1-CreERT2-eYFP; tdTomatoLSL mice demonstrates tamoxifen-dependent and transient expression of the tdTomato transgene with expression of tdTomato at 3, but not 24, d after tamoxifen treatment. D, Cx3CR1-CreERT2-eYFP; R26-tdTomato mice were generated, and sections from lumbar spinal cord demonstrate tdTomato expression that is tamoxifen-dependent and overlaps fully with Cx3CR1-eYFP, as well as the microglial marker, Iba1. Images taken at 20×, except 60× as indicated. Scale bar, 50 μm. E, TLR4 cKO scheme: crossing Cx3CR1-CreERT2-eYFP with TLR4fl/fl mice for several generations resulted in Cx3CR1-CreERT2-eYFP/+; TLR4fl/fl mice in which tamoxifen administration resulted in KO of TLR4 exon 3. F, Tlr4 mRNA expression was evaluated in whole brain by real-time RT-PCR and demonstrates persistent knockdown in CNS out to 28 d. There remains some Tlr4 mRNA in whole CNS tissue, likely ascribed to neuronal or astrocytic expression. n = 2-4 mice per group. **p < 0.01; ***p < 0.001 versus controls by one-way ANOVA with Tukey's post-test. G, Tlr4 mRNA expression was evaluated in spleen by real-time RT-PCR and demonstrates no significant decrease in tlr4 mRNA in spleen. H, Microglia were isolated from adult brain 3 weeks after tamoxifen using FACS (gated live cells: CD19– CD3– CD45mid CD11bhi Cx3CR1+) followed by real-time RT-PCR, which showed normal expression of tlr4 mRNA in the control and undetectable levels in the male and female TLR4 cKO samples. RNA gels shown on top with Gapdh as the housekeeping gene control and sample with no reverse transcription as the negative control. Fold change from control shown in graph with n = 2 mice per group. ***p < 0.001 versus control by one-way ANOVA with Tukey's post-test.