Fig 1: rAAV9 Transduces Osteoclast Lineage Cells In Vitro and In Vivo(A–D) A single dose of PBS or 8 × 1011 genome copies (GC) of rAAV.EGFP was intravenously (i.v.) injected into 2-month-old male mice, and EGFP expression was assessed in cryosectioned femurs by fluorescence microscopy 2 weeks post-injection. (A) Arrow indicates megakaryocytes with autofluorescence. TB, trabecular bone; BM, bone marrow; GP, growth plate (n = 3/group). Scale bars, 100 μm. (B) Cells were also immunostained with anti-CTSK antibody to identify osteoclast (OC)-lineage cells. Arrowheads indicate AAV9-transduced CTSK+ OCs. Scale bar, 75 μm. (C and D) Alternatively, EGFP expression in bone marrow cells was assessed by flow cytometry. Flow cytometry gating strategy of OC precursors (OCPs; CD3ε−, B220−, TER119−, CD11b−/lo, Ly6c+) is described in Figure S2C. GFP-expressing, CD11b-positive cells (C) and OCPs (D) are displayed in the dot plot and histogram, respectively. (E) Bone marrow-derived monocytes (BMMs) were cultured with M-CSF or GM-CSF (granulocyte-macrophage colony-stimulating factor) for 6 days to differentiate into bone marrow-derived macrophages (BMDMs) or dendritic cells (BMDCs), respectively. 1011 GC of rAAV9.EGFP were used to treat BMMs at day 0, or BMDMs and BMDCs at day 6, of culturing. Transduction efficiencies were assessed by EGFP expression using fluorescence microscopy. Cell nuclei were stained by DAPI. Scale bars, 1 mm. Alternatively, BMMs were cultured with M-CSF and RANKL for 2 and 6 days to differentiate into pre-OCs and mature OCs, respectively. rAAV9.EGFP was used to treat pre-OCs at day 2 or mature OCs at day 6 of culturing. (F and G) Two days after treatment with M-CSF and RANKL, Rankfl/fl pre-OCs were transduced with either rAAV9 carrying EGFP control (rAAV9.vec) or Cre recombinase (rAAV9.Cre) and then differentiated into mature OCs. Levels of (F) cre or rank mRNA and (G) TRAP activity were measured by RT-PCR (F) and colorimetric assay (G, left). Representative images of TRAP-stained OCs are displayed (G, right). Scale bars, 1 mm. (H–J) A single dose of 8 × 1011 GC of rAAV9.vec or rAAV9.cre was i.v. injected into 3-month-old female Rankfl/fl;RosamTmG mice. Fluorescence microscopy was performed on cryosectioned femurs to identify EGFP-expressing cells 2 weeks post-injection (H), and femoral trabecular bone mass was assessed by microCT 2 months post-injection. Representative 3D reconstruction (I) and relative quantification (J) are displayed. Trabecular bone volume/total volume (Tb.BV/TV), trabecular thickness (Tb.Th), trabecular number per cubic millimeter (Tb.N), and cortical thickness (Cort.Th) are shown (n = 5/group). Scale bars, 200 μm. Values represent mean ± SD. ∗p < 0.05, ∗∗∗p < 0.001 ∗∗∗∗p < 0.0001 by an unpaired two-tailed Student’s t test.