Extended Data Fig. 3: Generation and validation of Il3GFPfl/flAldh1l1CreERT25xFAD and Il3rafl/flCx3cr1CreERT25xFAD mice.
From: Astrocytic interleukin-3 programs microglia and limits Alzheimer’s disease
a, b, Schematics of the endogenous loci and editing strategies to generate conditional/reporter models for Il3 (a) and Il3ra (b). Mice were generated by excising large fragments of the endogenous loci by co-delivery of SpCas9 and two gRNAs, and in the presence of a long single-stranded DNA donor encoding a loxP-cDNA-P2A-EGFP-loxP cassette. c, d, Representative Sanger sequencing traces validating insertion of the loxP-cDNA-P2A-EGFP-loxP cassettes at the endogenous loci of Il3 (c) and Il3ra (d). Missense mutation quenches the GFP signal in Il3ra-targeted mice. e, GFP signal in ex vivo-stimulated splenic and lymph node T cells, known IL-3 sources, from WT and Il3GFPfl/fl mice. f, Flow cytometry analysis of astrocyte IL-3 production in 5-month-old Il3GFPfl/fl5xFAD and Il3GFPfl/flAldh1l1CreERT25xFAD mice injected with tamoxifen. g, qPCR analysis of Il3 mRNA expression in sorted astrocytes (n = 4 Il3GFPfl/fl5xFAD mice; n = 5 Il3GFPfl/flAldh1l1CreERT25xFAD mice). h, CSF IL-3 levels. Filled circles, males; open circles, females (n = 6 Il3GFPfl/fl5xFAD mice; n = 9 Il3GFPfl/flAldh1l1CreERT25xFAD mice). i, STAT5 phosphorylation in ex vivo heart macrophages from Il3rαfl/fl mice simulated with rIL-3. j, Flow cytometry analysis of microglia IL-3Rα production in 5-month-old Il3rαfl/fl5xFAD and Il3rαfl/flCx3cr1CreERT25xFAD mice injected with tamoxifen. k, qPCR analysis of Il3rα mRNA expression in sorted microglia (n = 4 Il3rαfl/fl5xFAD mice; n = 5 Il3rαfl/flCx3cr1CreERT25xFAD mice). All Il3GFPfl/fl5xFAD, Il3GFPfl/flAldh1l1CreERT25xFAD, Il3rαfl/fl5xFAD, and Il3rαfl/flCx3cr1CreERT25xFAD mice were injected with tamoxifen beginning at 2 months of age. **P < 0.01, ***P < 0.001, two-tailed Mann–Whitney U-tests. Mean ± s.e.m.
