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Yujin Zhang, Yasuhito Hayashi, Winston Kao, Chia-yang Liu; The generation and characterization of a novel mouse line, Keratocan- rtTA (KeraRki), for corneal research. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):1945.
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© ARVO (1962-2015); The Authors (2016-present)
To genetic modification of interested genes specifically in corneal stroma keratocytes during embryonic development and adult homeostasis, a novel keratocan-rtTA knock-in (KeraRki) mouse line was made in our libratory.
A gene-targeting construct containing an internal ribosomal entry site-reverse tetracycline transcription activator (IRES-rtTA) cassette was inserted into the keratocan gene to produce a knock-in mouse line via conventional gene targeting techniques. The resulting KeraRki mouse was first bred with FLP mice to remove Neo gene in the cassette. Then it was crossed to TetO-TH2-GFP (TH2) reporter mouse line to obtain KeraRki/TH2 bi-transgenic mice for revealing rtTA expressing cells with EGFP fluorescence after doxycline (Dox) administration. The EGFP fluorescence in KeraRki/TH2 was examined under a Zeiss stereomicroscope.
Ten chimeric mice were obtained after injection of six independent targeted ES clones into mouse blastocysts. Two chimeras (F0) consisting of 50% black coat color gave rise to the germline transmission and were used to mate with FLP mice. The Neo cassette was successfully removed in these two mouse clones carrying both KeraRki and FLP transgenes. When KeraRki/TH2 bi-transgenic mice were subjected to Dox induction from embryonic day 0 (E0) to postnatal day 1(P1), strong EGFP fluorescence labeled cells were specifically detected in corneal stroma and tendon of the limbs. However, there is no EGFP fluorescence in corneal epithelium or endothelium.
The novel KeraRki mouse line has been established that can drive expression of rtTA in corneal stroma and in tendon. This mouse will be a useful tool for gene manipulating and elucidating the biological functions in corneal stroma and limb during embryonic development, maintenance of homeostasis and pathogenesis. It can also be helpful in the analysis of neural-crest cell lineage.
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