Transgene construction, CSF-1 expression, and growth curves for transgenic mice. (A) Genomic organization of the mouse CSF-1 gene and transgenic constructs. Top diagram: CSF-1 gene showing intron-exon boundaries and the full-length coding sequence with positions of the glycosaminoglycan addition site (large arrowhead), the 3 major putative cleavage sites (small arrowhead) that generate sgCSF-1 (c1) and spCSF-1 (c2 or c3), and the transmembrane domain (TM). Bottom diagram: TgSPP and TgSGP transgenes, prepared by cloning the exons 2 to 9 fragment of the cDNA encoding a full-length CSF-1 downstream of the 3.13-kb CSF-1 promoter and first intron fragment. An additional hGH polyA signal fragment was added at the 3′ end of the cDNA. The exons 2 to 9 fragments used for both TgSGP and TgSPP contained a stop codon at amino acid 456 (diamond) to ensure that they were secreted. The fragment used for TgSGP also contained a mutation (S276L-G277A) in the unique glycosaminoglycan (chondroitin sulfate) addition site (SGXG/A). P1 and P2 indicate the oligonucleotide primer pairs used to detect the Csf1^op mutation in exon 4; P3 and P4, the primer pairs for amplifying a region between exons 2 to 4 to distinguish the transgene from the wild-type gene; and the exon 9 RPA probe was used to detect exon 9-containing mRNA expression. Relevant restriction sites are also indicated. Struck-out text indicates mutations of sites. (B) Fluorescence-activated cell sorter (FACS) analysis of cell-surface expression of CSF-1 in skin fibroblasts from transgenic lines. Expression profiles for wt, Csf1^op/Csf1^op; +/+ (filled), wt + URA (wt with unrelated antibody), Csf1^op/Csf1^op; TgSPP2/+, Csf1^op/Csf1^op; TgSPP7/+, Csf1^op/Csf1^op; TgSGP2/+, and Csf1^op/Csf1^op; TgSGP4/+ are shown. (C) Circulating CSF-1 concentrations in TgSPP and TgSGP mouse serum measured with a radioimmunoassay (RIA) that detects only biologically active CSF-1 and both spCSF-1 and sgCSF-1 equivalently (± SD, n > 5 for each line). +/+ indicates wild type; +/op, Csf1^op heterozygotes; and op/op, Csf1^op homozygotes. ^*P < .01 (compared with Csf1^op/+). (D) Nonreducing SDS-PAGE and anti-CSF-1 Western blot of immunoaffinity purified CSF-1 from conditioned media (lane 1, wt; lane 2, Csf1^op/Csf1^op; TgSPP/+; lane 3, Csf1^op/Csf1^op; TgSGP/+) and cell lysates (lane 4, Csf1^op/Csf1^op; TgCS/+) from immortalized skin fibroblasts. (E) Growth curves. Left panel: wt, Csf1^op/Csf1^op; TgSGP2/+, Csf1^op/Csf1^op; TgSGP4/+, Csf1^op/Csf1^op; +/+. Right panel: wt, Csf1^op/Csf1^op; TgSPP2/+, Csf1^op/Csf1^op; TgSPP7/+. ^*Significantly different from wt; P ≤ .05; n ≥ 6 mice at each time point.