Generation of an Iba1-EGFP Transgenic Rat for the Study of Microglia in an Outbred Rodent Strain

Flow cytometry validation of microglial Iba1-EGFP expression in neonates and adults. A, Brain cells isolated from either EGFP^+/+ (top) or wild-type littermates (bottom) at P7 were gated as EGFP⁺ and analyzed for their expression as CD11b⁺/CD45^int (right). Gated on forward scatter (FSC)/side scatter (SSC), singlets, live, CD45^int, CD11b⁺. B, The same samples from A were reanalyzed, gated as CD11b⁺/CD45^int (left) and analyzed for their expression as EGFP⁺ (right). In B and C, the black boxes indicate gates, and the percentage of cells within each gate are shown. C, Quantification of the median fluorescence intensity of EGFP⁺ cells isolated from heterozygous or homozygous littermates at P7. D, Brain cells isolated from either EGFP^+/+ (top) or wild-type littermates (bottom) at P60 or later were gated as CD11b⁺/CD45^int (left) and analyzed for their expression as EGFP⁺ (right). E, qPCR quantification of microglial genes (Iba1, CX3CR1) and nonmicroglial genes (Nes, Gfap, Olig2, NeuN) from EGFP⁺ sorted cells or whole-brain homogenates from EGFP^+/+ animals at P7. E, Brain cells from adult EGFP^+/+ and WT animals gated on CD11b⁺/CD45^int were analyzed for their expression as EGFP⁺. Histogram reflects a shift in EGFP fluorescence intensity in adult microglia-like cells in the Iba1-EGFP rat (green) compared with WT control (gray). Contour lines in A, B, and D represent 95% of the data at 5% intervals. Black boxes in A, B, and D indicate gates, and the percentage of cells within each gate are shown. Bars in C represent the mean ± SEM. Bars in E represent the mean ± SD. Open circles in C represent individual animal datapoints. *p < 0.05; **p < 0.01; ***p < 0.001.

Comparison of EGFP signal in blood and brain. A, Representative contour plots showing the gating strategy for median fluorescence intensity analysis in B and C. Cells were gated as EGFP⁺ and CD45⁺ in samples from blood (left) or brain (right) at P7 from either EGFP^+/+ (top) or wild-type littermates (bottom). B, Representative histograms showing the distribution of EGFP fluorescence intensity of all CD45⁺ cells at P7 in the EGFP^+/+ rat (green) compared with wild-type controls (gray) in the blood and brain. C, Quantification of median fluorescence intensity at P7. D, Representative contour plots showing the gating strategy for median fluorescence intensity analysis in E and F. Cells were gated as EGFP⁺ and CD45⁺ in samples from blood (left) or brain (right) in adults >P60 from either EGFP^+/+ (top) or wild-type littermates (bottom). E, Representative histogram showing the distribution of EGFP⁺ fluorescence intensity of all CD45⁺ cells in the adult EGFP^+/+ rat (green) compared with wild-type controls (gray) in the blood and brain. F, Quantification of median fluorescence intensity in adults at P60 or later. Contour lines in A represent 95% of the data at 5% intervals. Bars in C and F represent the mean ± SEM. Open circles represent individual animal datapoints.

Comparison of EGFP signal in spinal cord and dorsal root ganglion. A, Spinal cord cells isolated from either EGFP^+/+ (top) or wild-type littermates (bottom) at P7 were gated as EGFP⁺ (left) and then gated as CD11b⁺/CD45⁺ (right). B, The same samples from A were reanalyzed, gated as CD11b⁺/CD45⁺ (left), then gated for EGFP expression (right). C, Dorsal root ganglion cells isolated from either EGFP^+/+ (top) or wild-type littermates (bottom) at P7 were gated as EGFP⁺ (left) and then gated as CD11b⁺/CD45⁺ (right). D, The same samples from C were reanalyzed, gated as CD11b⁺/CD45⁺ (left), then gated for EGFP expression (right). Contour lines in A–C represent 95% of the data at 5% intervals. Black boxes in A–D indicate gates, and the percentage of cells within each gate are shown.

Flow cytometric characterization of EGFP⁺ peripheral cells in the adult. A, Quantification of the percentage of CD45^high peripheral immune cells expressing EGFP in the brain compared with the blood. B, Graph depicting the population subsets of CD45^high/EGFP⁺ cells in the brain and blood. (B cells = CD45^high, CD45R⁺; T cells = CD45^high, CD3⁺; myeloid cells/macrophages = CD45^high, CD11b⁺, RT1B⁺; monocytes = CD45^high, CD11b⁺, His48⁺; other = CD45^high, additional immune cells not determined by this panel). C, Pie charts showing the relative distribution of CD45^high/EGFP⁺ immune cells in the blood (left) and brain (right). D, Contour plots depicting the gating strategy for myeloid cells/macrophages in the brain (top) and blood (bottom). Quantification of the percentage of myeloid cells/macrophages expressing EGFP in the blood and brain. E, Contour plots showing the gating strategy for monocytes in the brain (top) and blood (bottom). Quantification of the percentage of monocytes expressing EGFP in the blood and brain. Contour lines (D, E) represent 95% of the data at 5% intervals. Bars represent the mean ± SD. Open circles and solid shapes represent individual animal datapoints. *p < 0.05; **p < 0.01; ***p < 0.001.

Histologic validation of knock-in efficiency. A, D, G, J, Representative coronal images from EGFP^+/− animals antibody labeled for Iba1 (red), GFP (green), and colabeled with DAPI (blue). White box shows the region analyzed for the prefrontal cortex (A–C), nucleus accumbens (D–F), hippocampus (G–I), and amygdala (J–L). B, E, H, K, Representative single-channel and merged images at 20× magnification. C, F, I, L, Quantification of the percentage of colocalization between GFP and Iba1 in heterozygous and homozygous animals. Bars represent the mean ± SEM. Open circles represent individual animal datapoints.