GFP expression in monocyte and macrophage populations. (A) The 2940-bp hCD68 promoter and intron 1 cassette were cloned upstream of EGFP and transgene transmission confirmed by PCR analysis of DNA from ear snips. (B) Confocal microscopy of a blood smear from a CD68-GFP mouse showed positive GFP expression in blood monocytes. (C) FISH analysis of metaphase chromosome preparations using a CD68 probe (red signal) and chromosome paint (green) made from mouse fibroblasts cultures counterstained with DAPI (blue). The green arrow indicates the transgene site integration on chromosome 3. (D) GFP mRNA expression was confirmed by RT-PCR analysis of tissue lysates from brain, lung, spleen, thymus, and liver of CD68-GFP and WT. (Dashed vertical line has been inserted to indicate a repositioned gel lane.) (E) Western blot analysis for GFP protein expression in tissue lysates (30 µg/tissue) from brain, lung, spleen, and thymus of CD68-GFP and WT mice. (F) To determine transgene copy number, 100 ng of DNA was isolated from the thymus of male CD68-GFP and male WT mice, amplified using standard PCR, and run on agarose gel side-by-side with the pEGFP-N1 vector. A male GFP knock-in (KI) mouse that carries a single copy of the GFP transgene on the X chromosome and a female homozygote GFP KI mouse that carries 2 copies of the GFP transgene on the X chromosome were used as copy number controls. (G) Transgene copy number was quantified with quantitative PCR using a 10-fold serial dilution standard curve of the pEGFP-N1 vector. (H) Table shows mean relative copy number ± SEM.