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L. Wang, H. Liu, W.W. Y. Kao; Tet–On Mouse Lines for Cornea–Epithelium Specific Gene Ablation . Invest. Ophthalmol. Vis. Sci. 2006;47(13):1586.
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Conventional gene targeting strategy is a useful technique to create mouse lines in which the genes of interest are ablated (knockout) or modified (knock–in) for determining functions of genes. However, embryonic lethality and congenital defects often prevent the use of such genetically modified mice to further study the roles of such genes in pathogenesis of acquired diseases, e.g., wound healing, inflammation, immune disorders, etc. To overcome this limitation, we examined the efficacy of a trigenic K12rtTA/w/tet–O–Cre/ZEG mouse line that expresses EGFP (Enhanced Green Fluorescent Protein) upon doxycycline induction.
We previously created a Krt12rtTA/rtTA mouse line that constitutively synthesizes rtTA (reverse tetracycline transcription activator: tet–ON) by corneal epithelium via a knock–in strategy of gene targeting technique (Chikama et al, IOVS 46:1966–1972, 2005). ZEG is a reporter transgenic mouse line created with a transgene consisting of a chicken actin promoter, a LacZ gene flanked by two loxP elements and followed by an EGFP minigene, tet–O–Cre transgenic mouse carries a TRE–Cre minigene (tetracycline responsive elements). Trigenic K12rtTA/w/tet–O–Cre/ZEG mice were obtained by crossbreeding Krt12rtTA/rtTA, tet–O–Cre and ZEG mice. Adult trigenic mice were fed doxycycline for one week and then fed regular chow for four more weeks. ZEG and tet–O–Cre/ZEG transgenic mice were used as controls. The expression of EGFP was determined by a ZEISS fluorescent stereomicroscope (model SV11).
The trigenic mice did not express green fluorescence protein in the absence of doxycycline induction. Upon induction by doxycycline, green fluorescence was detected in corneas of trigenic mice fed doxycycline for one day, the green fluorescence intensity increased and reached a plateau in seven days. The green fluorescence diminished when doxycycline was removed from the diet and vanished in four weeks. The control ZEG and tet–O–Cre/ZEG mice did not express EGFP.
The expression of Cre recombinase can be induced in corneas of trigenic K12rtTA/w/tet–O–Cre/ZEG mice, which excises the floxed LacZ and leads to the expression of EGFP. This strategy of breeding K12rtTA/w/tet–O–Cre/Xf/f will allow us to create mouse lines in which the floxed genes can be ablated in corneal epithelium upon doxycycline induction
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