Figure 2.

Structure of the CD11b-DTR transgene and ablation specificity. (A) Diagram representing the MacT6 construct. The cDNA for the DTR-eGFP fusion gene is inserted between the human CD11b promoter (coordinates –1,704 to +83 relative to transcription start) and human growth hormone (hGH) sequence that provides splicing and polyadenylation sequences. Small half-arrows indicate location of the nucleotide pair used for RT-PCR transcript detection; right-facing arrow, transcription start point; gray boxes, exons; and open boxes, untranslated regions. RT-PCR analysis performed on BM-derived macrophages for the control F4/80 (B) and transgenic DTR-eGFP (C) transcripts. Results shown for WT mice and CD11b-DTR lines 34 (positive-line example) and 35 (negative-line example). The eGFP-hGH oligonucleotide pair specifically amplified the transgene mRNA; amplification products were not evident in samples from WT mice or when reverse transcriptase (RT) was omitted. (D–G) FACS assessment of peritoneal and spleen populations. (D) In WT mice, injection of 2 doses of DT at 25 ng/g mouse weight does not affect either the small population of CD3⁺ T cells (upper left quadrant) or the larger population of F4/80⁺ macrophages (lower right quadrant) in the peritoneal cavity. (E) In heterozygous CD11b-DTR mice receiving DT, the F4/80⁺ population is eliminated while the CD3⁺ cells remain. Neither the CD3⁺ nor the B220⁺ cells in the spleen were affected by DT injection either in WT (F) or CD11b-DTR mice (G). Wright-Geimsa–stained cytospin preparations of BTG-elicited peritoneal cells after DT^mut (H) or DT (I) injection into CD11b-DTR mice. Normal proportions of elicited peritoneal cells were observed with injection of DT^mut, but the absence of macrophages and preponderance of neutrophils was apparent in mice treated with DT.