A 4kb Fragment of the Desmocollin 3 Promoter Directs Reporter Gene Expression to Parakeratotic Epidermis and Primary Hair Follicles  Anita J. Merritt, Kuichun Zhu, David R. Garrod, Martyn A.J. Chidgey  Journal of Investigative Dermatology  Volume 127, Issue 1, Pages 245-247 (January 2007) DOI: 10.1038/sj.jid.5700483 Copyright © 2007 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 β-Galactosidase is expressed in parakeratotic, but not orthokeratotic, epidermis. (a) Dsc3 5′-flanking DNA (4kb; from HindIII to base −1) was cloned into the EcoRI and XhoI sites of pNASSβ (Clontech, Basingstoke, Hampshire, UK), a promoterless eucaryotic expression vector containing an SV40 splice donor/splice acceptor (SD/SA), β-galactosidase coding sequence, and SV40 polyadenylation site. The Dsc3-βgal cassette was excised from the plasmid by cutting with KasI (partial) and SalI. DNA was recovered using the QIAEXII kit (Qiagen, Crawley, West Sussex, UK) and diluted in microinjection buffer (5mm Tris, 0.1mm EDTA, pH 7.4). Transgenic mice were generated as described (Merritt et al., 2002), with ethical approval from the Universities of Birmingham and Manchester, and under licence from the UK Home Office. (b) Mice carrying integrated transgene DNA were identified by Southern blotting of BamHI cut genomic DNA using an 827bp SacI–EcoRV probe from the β-galactosidase gene. tg, transgenic; wt, wild type. (c) Mouse tail showing β-galactosidase activity in scale epidermis but not in interscale regions. Tissues from transgenic mice were fixed for 30minutes in 0.2% glutaraldehyde, 0.1m sodium phosphate (pH 7.3), 5mm EGTA, and 2mm MgCl2, washed (3 × 30minutes) with 0.1m sodium phosphate (pH 7.3), 2mm MgCl2, 0.01% sodium deoxycholate, and 0.02% IGEPAL CA-630, and stained overnight at 37°C in wash buffer with 5mm potassium ferrocyanide, 5mm potassium ferricyanide, and 1mg/ml 5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside (X-gal). Stained tissues were washed, post-fixed in formal saline, embedded in paraffin, sectioned, and stained with eosin. Arrow indicates anterior to posterior direction. (d) Transverse section of tail showing β-galactosidase activity in scale epidermis but not in interscale regions. β-Galactosidase activity is also associated with primary hair follicles in the tail (arrowheads). Note the characteristic presence of three hair follicles associated with each scale. sr, scale region; isr, interscale region. (e) Tail epidermis showing Dsc3 native protein expression (brown immunostaining) in the epidermis. The streptavidin–biotin indirect immunoperoxidase method was used with a primary rabbit anti-mouse Dsc3 antibody (Hardman et al., 2005) at a 100-fold dilution. Antigen retrieval was performed by microwaving (3minutes in 0.01m citrate buffer, pH 6) and the primary antibody was conjugated with biotinylated goat anti-mouse/rabbit IgG (Dako, Ely, Cambridgeshire, UK) and streptavidin–peroxidase. Sections were stained with 3,3′-diaminobenzidine (Sigma, Poole, Dorset, UK) and counterstained with hematoxylin. Dsc3 is expressed in both scale and interscale epidermis. (f) High magnification of scale epidermis showing absence of β-galactosidase activity in the basal cell layer (arrowhead). (g) High magnification of scale epidermis showing Dsc3 expression in all cell layers, including the basal cell layer (arrowhead). (h) Expression of β-galactosidase in all cell layers of scale epidermis of leg. Staining is absent from interscale epidermis. The basal layer is indicated by an arrowhead. (i) β-Galactosidase activity in developing scales and primary hair follicles (arrowheads) of an 8-day mouse. Bar=100μm in (d), (e), and (i) and 25μm in (f), (g), and (h). Journal of Investigative Dermatology 2007 127, 245-247DOI: (10.1038/sj.jid.5700483) Copyright © 2007 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 β-Galactosidase is expressed in the bulb region of primary hair follicles. (a) Dsc3 and (b) β-galactosidase expression in the bulb of a primary hair follicle from tail. Arrowheads indicate specific Dsc3 staining at the cell membrane. Bar=10μm. Journal of Investigative Dermatology 2007 127, 245-247DOI: (10.1038/sj.jid.5700483) Copyright © 2007 The Society for Investigative Dermatology, Inc Terms and Conditions