1. Conditional Gene Expression in the Epidermis of Transgenic Mice Using the Tetracycline-Regulated Transactivators tTA and rTA Linked to the Keratin 5 Promoter 
Ilysa Diamond, Timothy Owolabi, Melissa Marco, Christopher Lam, Adam Glick 
Journal of Investigative Dermatology 
Volume 115, Issue 5, Pages (November 2000)
DOI: /j x
Copyright © 2000 The Society for Investigative Dermatology, Inc Terms and Conditions

2. Figure 1
Generation of K5/tTA and K5/rTA transgenic lines. (A) Diagram of the constructs used for microinjection. An EcoR1-BamH1 fragment containing the tTA or rTA gene was inserted into the polylinker of the plasmid pBK5-97, which contains 5.2 kb from the bovine K5 promoter (Ramirez et al. 1994), a β-globin intron, and SV40 poly-A addition sequence. (B) Southern blot analysis of founder lines for the presence of tTA transgene by hybridization to tTA probe. The autoradiograph shows all tTA founders (1196–1156), of which only line 1216 transactivated in vivo, and five of 21 rTA lines (A10-K4), all of which transactivated in vivo. (C) Northern blot analysis of tTA and rTA expression in the different founder lines. Total RNA from line 1216 (tTA) and A10-K4 (rTA) was hybridized to a TA-specific probe. The PL7 ribosomal protein cDNA was used to normalize loading. For each line, the level of TA expression was normalized to PL7 using scanning densitometry, and then expressed relative to that of line 1216 as indicated underneath each lane.
Journal of Investigative Dermatology  , DOI: ( /j x)
Copyright © 2000 The Society for Investigative Dermatology, Inc Terms and Conditions

3. Figure 2
β-Galactosidase expression in transgenic mice expressing K5/tTA or K5/rTA is regulated in a tissue-specific manner. (A) Tissue-specific transactivation of β-galactosidase is suppressed by doxycycline in K5/tTA × tetOlacZ double transgenic mice. (B) Tissue-specific transactivation of β-galactosidase is induced by doxycycline in K5/rTA × tetOlacZ double transgenic mice. Levels of β-galactosidase enzyme activity were determined in tissue extracts from double and single transgenic mice obtained from crosses of the K5/tTA and different K5/rTA founders with the tetOlacZ line. To induce or suppress transactivation, mice were placed on feed containing 1 g per kg doxycycline. Each bar represents the average of two to three mice ± SEM.
Journal of Investigative Dermatology  , DOI: ( /j x)
Copyright © 2000 The Society for Investigative Dermatology, Inc Terms and Conditions

4. Figure 3
In situ analysis of β-galactosidase expression in K5/tTA and K5/rTA double transgenic mice. Histochemical detection of β-galactosidase enzyme activity in tissues of double transgenic mice. β-Galactosidase enzyme activity was assayed in situ as described in Materials and Methods in frozen tissue sections from K5/tTA × tetOlacZ double transgenic (A, D) or K5/rTA × tetOlacZ double transgenic (B, C, E-G) mice in the absence (A, E) or presence (B–D, F, G) of 1 g per kg doxycycline. Tissues shown are dorsal epidermis (A-E), tongue (F), and forestomach (G). Part (B) represents the β-galactosidase expression pattern in the epidermis obtained with mice from founder line B1; part (C) represents the expression pattern obtained with mice of lines F6, K4, A10, and G5.
Journal of Investigative Dermatology  , DOI: ( /j x)
Copyright © 2000 The Society for Investigative Dermatology, Inc Terms and Conditions

5. Figure 4
Suppression and reexpression of β-galactosidase in K5/tTA × tetOlacZ mice is dependent on doxycycline dose. (A) Dose- dependent suppression of β-galactosidase activity by oral doxycycline. Double transgenic mice were dosed for 20 d with varying concentrations of doxycycline in the drinking water with 5% sucrose, and β-galactosidase enzyme activity was measured in skin extracts as described in Materials and Methods. Data are plotted as percentage of enzyme activity in untreated double transgenic animals. Each dose is the average obtained from two animals. (B) Time course of β-galactosidase induction following removal of oral doxycycline. Double transgenic mice were put on the indicated doses of doxycycline in the drinking water for 20 d, and skin biopsies were taken to measure suppressed levels of β-galactosidase enzyme activity (day 0). Mice were then put on deionized water and skin biopsies were taken 1, 2, 3, and 8 d after withdrawal from doxycycline. Each bar represents the average activity in extracts from two to three mice. ST, single transgenic; DT, double transgenic.
Journal of Investigative Dermatology  , DOI: ( /j x)
Copyright © 2000 The Society for Investigative Dermatology, Inc Terms and Conditions

6. Figure 5
Doxycycline causes a rapid induction of β-galactosidase activity in the epidermis of K5/rTA × tetOlacZ double transgenic mice. (A) β-Galactosidase levels increase in the skin of double transgenic mice within 24 h after administration of doxycycline. β-Galactosidase levels were determined in skin extracts from mice maintained on regular feed, and at the indicated time points after switching to 1 g per kg doxycycline feed. Each bar represents the average of three mice. (B) Histochemical detection of β-galactosidase enzyme activity in the hair follicles of double transgenic mice 24 h after administration of doxycycline. Scale bar: 1 μm.
Journal of Investigative Dermatology  , DOI: ( /j x)
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7. Figure 6
Topical doxycycline suppresses β-galactosidase gene expression in the epidermis of K5/tTA × tetOlacZ double transgenic mice. (A, B) Histochemical detection of β-galactosidase activity in the skin of double transgenic mice treated topically with doxycycline. Doxycycline in 40% ethanol or vehicle alone was applied to the shaved back of double transgenic mice, and the skin was isolated after 24 h for in situ histochemical detection of β-galactosidase activity as described in Materials and Methods. (A) -dox and (B) +dox sections were stained for β-galactosidase for the same length of time. hf, hair follicle. Arrow indicates the basal layer of the epidermis showing decreased levels of β-galactosidase activity in (B) compared with (A). (C) Suppression of β-galactosidase mRNA levels by topical doxycycline. Semiquantitative RT-PCR analysis of β-galactosidase mRNA levels in double transgenic (DT) or single transgenic (ST) mice treated topically for 24 h with vehicle (–) or 1 μg per ml doxycycline (+). Each lane represents an individual mouse.
Journal of Investigative Dermatology  , DOI: ( /j x)
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8. Figure 7
TPA treatment causes rapid migration of cells out of the hair follicle. (A) K5/rTA line F6 × tetOlacZ double transgenic mice were placed on 1 g per kg doxycycline chow for 1 wk. TPA (10 μg in 200 μl acetone) was applied to the shaved dorsal surface of the mice and skin biopsies were taken at 24, 36, and 48 h and stained for β-galactosidase. No β-galactosidase staining was observed in the epidermis of mice prior to TPA treatment, or with acetone treatment alone. Similar results were obtained with three different mice treated with TPA. (B) TPA does not induce expression of the K5/rTA transgene. Total RNA was isolated from transgenic mice 36 h after treatment with TPA or acetone as above, and hybridized to a TA-specific hybridization probe. Northern blots were stripped and rehybridized to PL7 to control for loading.
Journal of Investigative Dermatology  , DOI: ( /j x)
Copyright © 2000 The Society for Investigative Dermatology, Inc Terms and Conditions