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Tai-ichiro Chikama, Yasuhito Hayashi, Chia-Yang Liu, Noriko Terai, Kazuto Terai, Candace W.-C. Kao, Li Wang, Miyuki Hayashi, Teruo Nishida, Philip Sanford, Tom Doestchman, Winston W.-Y. Kao; Characterization of Tetracycline-Inducible Bitransgenic Krt12rtTA/+/tet-O-LacZ Mice. Invest. Ophthalmol. Vis. Sci. 2005;46(6):1966-1972. doi: 10.1167/iovs.04-1464.
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purpose. To prepare binary transgenic mouse lines that overexpress reporter genes in a corneal-epithelium–specific manner when induced by doxycycline.
methods. A gene-targeting construct containing an internal ribosomal entry site–reverse tetracycline transcription activator (IRES-rtTA) cassette was inserted into the Krt12 allele (keratin 12 gene) to produce a knock-in Krt12 rtTA/+ mouse line through gene-targeting techniques. The Krt12 rtTA/+ knock-in mice were bred with tet-O-LacZ reporter mice to obtain Krt12 rtTA/+ /tet-O-LacZ bitransgenic mice. The expression of the LacZ gene was induced in bitransgenic mice by administration of doxycycline in the drinking water and chow.
results. Administration of doxycycline induced a 15-fold increase of β-galactosidase activity in the cornea of adult bitransgenic mice (Krt12 rtTA/+ /tet-O-lacZ). Administration of doxycycline either to single transgenic Krt12 rtTA/+ or tet-O-LacZ mice as a control did not induce overexpression of LacZ as it did in the bitransgenic mice. The induction of β-galactosidase enzyme activity by doxycycline in bitransgenic mice took place in 24 hours and reached a plateau by 2 days. Histochemical analysis also showed that β-galactosidase induction was limited to the corneal epithelium of bitransgenic mice fed doxycycline. The increased β-galactosidase activity in corneal epithelium caused by doxycycline returned to basal levels in 4 weeks after the antibiotics were omitted from the diet.
conclusions. A binary mouse model has been successfully established that conditionally overexpresses reporter genes in corneal epithelium. This mouse model will be useful in elucidating signaling pathways of various growth factors and cytokines and gene functions in the maintenance of homeostasis and pathogenesis in the adult mouse cornea.
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