Journal: Nature Communications
Article Title: A lung tropic AAV vector improves survival in a mouse model of surfactant B deficiency
Figure Lengend Snippet: AAV6.2FF transduces both airway and alveolar epithelial cells. a Whole-mount X-gal staining of the lungs, nasal cavity and trachea 3 weeks post-vector administration from either 1 × PBS or AAV6.2FF-emGFP-nlsCre treated Rosa26-Flox/LacZ mice. b Representative nuclear fast red counterstained paraffin sections (4 μm) from X-gal stained lungs. LacZ-positive cells were found in the distal airway epithelium and alveoli following delivery of AAV6.2FF-CMV-emGFP-nlsCre. Morphologic criteria demonstrate that both AT2 (white arrows) and club cells (yellow arrow) are X-gal positive (Distal lung scale bars represent 100 μm, 50 μm, and 20 μm from left to right; Trachea scale bars represent 200 μm, 50 μm, and 20 μm from left to right). c 3D histograms from flow cytometry analysis of dissociated whole lungs harvested from transgenic SP-B mice 8 days after IT administration of 10 11 vg per mouse of AAV-Luc control ( n = 2), or 10 11 vg per mouse of AAV6.2FF-GFP (AAV-GFP; n = 4). The first set of histograms represent the total CD45 negative, EpCAM++ (high expressing) cell population (first peak), and the CD45 negative, EpCAM++ cells that are GFP positive (second peak) in each mouse. The second set of histograms are the rescaled CD45 negative, EpCAM++, GFP positive cell populations as indicated by the black box in the first histogram. The column graph represents the mean percentage of total CD45 negative, EpCAM++ cells that are GFP positive with SD ( P value = two-tailed Student’s t test, * P = 0.0127). d This column graph represents the mean percentage of different lung cell populations with SD that are GFP positive. CD45 positive staining represents hematopoietic cells, CD45 negative, EpCAM dim staining represents alveolar type 1 (AT1) epithelial cells, CD45 negative, EpCAM++ staining represents AT2 or ciliated cells (red circles and red box), CD45 negative, CD31 positive staining represents endothelial cells, and CD45 negative, EpCAM negative, CD31 negative represents all other cell types found in dissociated lung tissue ( n = 4 biologically independent animals per group). The source data for c and d have been provided as a Source Data file.
Article Snippet: Following perfusion, an enzyme mix containing 30 U neutral protease (Worthington; LS02104), 2500 U Collagenase I (Worthington; LS004196), 10 μg DNase I (Sigma; D5025-150KU) in DPBS with Mg2+ and Ca2+ was instilled into the lungs.
Techniques: Staining, Plasmid Preparation, Mouse Assay, Flow Cytometry, Transgenic Assay, Expressing, Two Tailed Test