Figure 6. Inter-loxP distance is a determinant of recombination efficiency in microglial CreER lines.

(A) Diagram of the Rosa26^Ai9 allele and Rosa26^mTmG 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 Rosa26^Ai9 and Rosa26^mTmG in microglia by flow cytometry.

(C, D, F, and G) Representative flow cytometry results from individual oil- and TAM-injected Cx3cr1^{YFP–CreER/+ (Litt)} and Tmem119^CreER/+ mice expressing Rosa26^Ai9/+ are compared with flow cytometry results of Rosa26^mTmG/+ from Figure 1. Recombined Rosa26^Ai9 microglia are tdTomato⁺, whereas recombined Rosa26^mTmG microglia are mGFP⁺ (mG) and non-recombined Rosa26^mTmG microglia are mTomato⁺ (mT).

(E and H) Quantifications of recombination of Rosa26^Ai9 and Rosa26^mTmG in microglia from oil- and TAM-injected mice show significantly more recombination of the 0.9 kb Rosa26^Ai9 floxed allele vs. the 2.4 kb Rosa26^mTmG floxed allele in (E) Cx3cr1^{YFP–CreER/+ (Litt)} mice (two-way ANOVA with Sidak’s post hoc test; n = 4 Rosa26^Ai9 oil, 5 Rosa26^mTmG oil, 4 Rosa26^Ai9 TAM, and 5 Rosa26^mTmG mice; ****p < 0.0001) and (H) Tmem119^CreER/+ mice (two-way ANOVA with Sidak’s post hoc test; n = 4 Rosa26^Ai9 oil, 8 Rosa26^mTmG oil, 4 Rosa26^Ai9 TAM, and 7 Rosa26^mTmG 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 Cx3cr1^{YFP–CreER/+ (Litt)} and Tmem119^CreER/+ mice expressing Rosa26^Ai9/+ or Rosa26^mTmG/+.

All data are presented as mean ± SEM. Individual data points indicate males (squares) and females (circles). See also Figure S6.