Induction of Corneal Epithelium–specific LacZ Expression in Bitransgenic Krt12–rtTA/tetOlacZ Mice

Abstract

Abstract: : Purpose: To prepare binary transgenic mouse lines that over express reporter genes in a corneal epithelium–specific manner upon induction by Doxycycline . Methods: A gene targeting construct containing internal ribosomal entry site–reverse tetracycline transcription activator (IRES–rtTA) cassette was inserted into Krt12 allele to produce a knock–in Krt12–rtTA mouse line via gene targeting techniques The Krt12^rtTA/+ knock–in mice were cross bred with reporter mouse line tet–O–LacZ mice to obtain Krt12^rtTA/+/tet–O–LacZ bitransgenic mice. The expression of LacZ gene was induced in bitransgenic mice by administration of doxycycline via drinking water and chow. Results:Adminstration of doxycycline caused a 15–fold increase of ß–galactosidase enzyme activity in the cornea of adult bitransgenic mice (Krt12^rtTA/+/tet–O–lacZ) above that of control without feeding the antibiotics, but not in other tissues. Administration of doxycycline to single transgenic Krt12^rtTA/+ and tet–O–LacZ mice as control did not induce over expression of LacZ in the experimental mice. The induction of ß–galactosidase enzyme activity by doxycycline in bitransgenic mice took place in 24 h and reached a plateau by 7 days. Histochemical analysis also showed ß–galactosidase induction was limited to the corneal epithelium of bitransgenic mice fed doxycycline. The increased ß–galactosidase activity in corneal epithelium caused by doxycycline returned to a basal leveling in 4 weeks when the antibiotics was omitted in the diet. Conclusions: We have successfully established a binary mouse model to conditionally over expressing reporter genes in corneal epithelium. This mouse model is useful to elucidate signaling pathways and function of gene of interest in maintenance of homeostasis and pathogenesis in adult mice.

Keywords: cornea: epithelium • gene/expression • transgenics/knock-outs