Figure 1
Design and validation of c-fms-eGFP-L10a construct and transgenic MacTRAP mice. (A) Transgene construction. A cassette encoding eGFP fused to the ribosomal protein L10a was inserted downstream of the c-fms promoter/enhancer element. (B) In vitro validation of the construct by fluorescence microscopy. RAW 264.7 cells were transfected with peGFP (control) or the c-fms-eGFP-L10a plasmid; latter reveal a nucleolar fluorescence signal which is in accordance with the site of ribosome assembly. By contrast, control cells show a homogenous fluorescence pattern throughout the cell body. (C,D) RT-qPCR of eGFP-L10a and endogenous Csf1r expression in various organs of MacTRAP mice. Mean ± SEM; n = 5. (E–G) Correlation between expression levels of the transgene c-fms-eGFP-L10a and endogenous Csf1r (r = 0.93, p < 0.01), F4/80 (r = 0.93, p < 0.01) and CD68 (r = 0.86, p < 0.05), respectively, across different organs of MacTRAP mice; data represent mean expression values ± SEM; n = 5. (H) Western Blot of spleen lysates from MacTRAP mice. Anti-GFP antibody detects a band at ~50 kDa, consistent with the expected size of the eGFP-L10a fusion protein. GAPDH served as loading control. Kidney lysates of PodoTRAP mice21 and WT mice served as positive control and negative control, respectively (full-length blot is shown in Sup. Fig. S10).
