Targeting expression of a transgene to the airway surface epithelium using a ciliated cell-specific promoter  Lawrence E Ostrowski, James R Hutchins, Kelly Zakel, Wanda K O'Neal  Molecular Therapy  Volume 8, Issue 4, Pages 637-645 (October 2003) DOI: 10.1016/S1525-0016(03)00221-1 Copyright © 2003 The American Society of Gene Therapy Terms and Conditions

FIG. 1 (A) Diagram of the FOXJ1 promoter construct used to produce transgenic animals. The promoter fragment consists of 1008 bp of the FOXJ1 genomic region (−80 to −1088 relative to the ATG) and includes the RNA transcription start site (−216) and a CpG island (−643 to −877). The construct also includes portions of two noncoding exons (shaded boxes, I and II) and the first intron from the mouse transthyretin (TTR) gene. The relative positions of the EGFP cDNA and the SV40 poly(A) site are indicated. The total size of the injected construct was 3.2 kb. (B) Southern analysis of FOXJ1/EGFP founder animals. DNA isolated from transgenic animals was probed for the presence of the EGFP transgene. Animals 45, 46, 49, and 85 were bred to establish four separate lines expressing EGFP from the FOXJ1 promoter. Molecular Therapy 2003 8, 637-645DOI: (10.1016/S1525-0016(03)00221-1) Copyright © 2003 The American Society of Gene Therapy Terms and Conditions

FIG. 2 EGFP fluorescence in frozen sections from FOXJ1/EGFP transgenic and wild-type mice. EGFP is clearly expressed in the ciliated epithelium of tracheal, lung, and nasal tissue from the transgenic animals (A, B, C), while sections from esophagus and muscle (D, E) and from wild-type animals (K, L, M, N, and O) show no EGFP fluorescence. (F, G, H, I, J, P, Q, R, S, and T) Adjacent sections stained with hematoxylin and eosin. All images were from line 45, except B, G, L, and Q, which were from line 49. Scale bars, 100 μm. Molecular Therapy 2003 8, 637-645DOI: (10.1016/S1525-0016(03)00221-1) Copyright © 2003 The American Society of Gene Therapy Terms and Conditions

FIG. 3 EGFP fluorescence in nonrespiratory tract tissues from FOXJ1/EGFP transgenic mice. (A) Ciliated ependymal cells lining the ventricles of the brain are strongly EGFP positive. Ciliated epithelium of the oviduct (B) and developing sperm in the testis (C) also express EGFP from the FOXJ1 promoter. Scale bars, 50 μm. A and B, line 45; C line 85. Molecular Therapy 2003 8, 637-645DOI: (10.1016/S1525-0016(03)00221-1) Copyright © 2003 The American Society of Gene Therapy Terms and Conditions

FIG. 4 Analysis of FOXJ1-driven EGFP expression in four different transgenic lines. (A) Frozen sections were prepared from trachea, lung, and esophagus from each of the indicated lines. Images were then captured using the same exposure conditions. All four lines examined showed similar patterns and intensities of EGFP fluorescence. Scale bar, 50 μm for all images. (B) Tracheal extracts were prepared from each of the four transgenic lines and wild-type littermate controls and analyzed by Western blotting. EGFP is present at varying levels in the transgenic lines and was not detected in the wild-type animals. Blots were reprobed with an antibody to βIV tubulin as a control for the amount of ciliary protein loaded. The level of EGFP expressed appeared independent of the transgene copy number. Molecular Therapy 2003 8, 637-645DOI: (10.1016/S1525-0016(03)00221-1) Copyright © 2003 The American Society of Gene Therapy Terms and Conditions

FIG. 5 Localization of EGFP expression by immunochemistry. Paraffin-embedded sections from FOXJ1/EGFP transgenic (A, B, C, D, E, G, I, and J) and wild-type (F, H, and K) animals were immunostained with the anti-GFP antibody, except that the section in C was probed with control rabbit IgG. EGFP was detected in ciliated cells of the trachea (A, B), conducting airways (D, E), and nasal epithelium (G, I, J) of the transgenic animals, while no specific staining was observed in the same tissues from wild-type animals. All animals were from line 85. Scale bars, A and C, 30 μm; D, F, I, and K, 50 μm; B, E, and J, 10 μm. Molecular Therapy 2003 8, 637-645DOI: (10.1016/S1525-0016(03)00221-1) Copyright © 2003 The American Society of Gene Therapy Terms and Conditions

FIG. 6 Localization of EGFP expression in ciliated cells identified by βIV tubulin immunostaining and confocal microscopy. Thick sections of a trachea from a FOXJ1/EGFP transgenic animal were immunostained with an anti-βIV tubulin antibody (detected with Texas red secondary) and examined by fluorescence confocal microscopy. Two different images are shown (A, B). EGFP expression is clearly restricted to the ciliated cells. Animal is from line 49. Scale bar, 10 μm. Molecular Therapy 2003 8, 637-645DOI: (10.1016/S1525-0016(03)00221-1) Copyright © 2003 The American Society of Gene Therapy Terms and Conditions