Figure 5. A qPCR protocol to quantitatively assess recombination in microglia.

A Diagram of protocol used to quantify Cre/LoxP recombination of microglial genomic DNA (gDNA) by quantitative PCR (qPCR). B Diagram of experiment to assess Cre/LoxP recombination in primary microglia cultures from Rosa26^mTmG/+; Cx3cr1^{YFP-CreER/+ (Litt)} mice after exposure to 4-hydroxytamoxifen (4-OHT). C Fluorescent images of endogenous mGFP (green) and endogenous mTomato (red) in primary microglia cultures from Rosa26^mTmG/+; Cx3cr1^{YFP-CreER/+ (Litt)} mice after exposure to 4-OHT. Scale bars=25 µm. D Flow cytometry analysis of recombined mGFP⁺ (mG) vs. non-recombined mTomato⁺ (mT) microglia in primary microglia cultures from Rosa26^mTmG/+; Cx3cr1^{YFP-CreER/+ (Litt)} mice after exposure to 4-OHT. E Quantification of the percentage of recombined gDNA by qPCR shows increased recombination in primary microglia exposured to 10 nM, 100 nM, or 1000 nM of 4-OHT (1-way ANOVA with Dunnett’s post-hoc; vehicle vs. 10 nM, n = 2 independent cultures, P < 0.0001, q = 60.37, df = 4; vehicle vs. 100 nM, n = 2 independent cultures, P < 0.0001, q = 70.21, df = 4; vehicle vs. 1000 nM, n = 2 independent cultures, P < 0.0001, q = 58.14, df = 4). F Graph of percent recombination of Rosa26^mTmG in primary microglia from Rosa26^mTmG/mTmG; Cx3cr1^{YFP-CreER/+ (Litt)} mice after exposure to 4OH tamoxifen as measured by flow cytometry analysis vs. the recombination rate as measured by qPCR of microglial gDNA isolated by fluorescence-activated cell sorting (FACS). Data points fit to a linear curve (black line; r² = 0.9540), closely aligned with the line of identity (red dashed line), indicating that qPCR provides a linear, quantitative measurement of Rosa26^mTmG recombination in in vitro samples. G Diagram of experiment to assess Cre/LoxP recombination in mice injected with tamoxifen (TAM) or oil. H Quantification of the percentage of recombined gDNA by qPCR shows increased recombination in TAM vs. oil for all three CreER lines (Rosa26^mTmG/+; Cx3cr1^{YFP-CreER/+ (Litt)}: Student’s t-test, n = 4 mice, P < 0.0001, t = 21.38, d f= 6; Rosa26^mTmG/+; Cx3cr1^{CreER/+ (Jung)}: Student’s t-test, n = 4 mice, P = 0.0003, t = 7.26, df = 6; Rosa26^mTmG/+; Tmem119^CreER/+: Student’s t-test, n = 4 oil, 3 TAM mice, P = 0.0111, t = 3.925, df = 5). I Graph of percent recombination of Rosa26^mTmG in microglia in mice injected with TAM or oil as measured by flow cytometry analysis (see also Fig. 1) vs. the recombination rate as measured by qPCR of microglial gDNA isolated by FACS. Data points fit to a linear curve (black line; r² = 0.9267), closely aligned with the line of identity (red dashed line), indicating that qPCR provides a linear, quantitative measurement of Rosa26^mTmG recombination in in vivo samples. Data in e and h are presented as mean ± SEM.