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Jianhua Zhang, Fei Dong, Yujin Zhang, Chia-Yang Liu, Yueh-Chiang Hu, Winston W Y Kao; Characterization of tet-On Krt4-rtTA Driver Mice Created via CRISPR/Cas9-mediated Knock-in Strategy. Invest. Ophthalmol. Vis. Sci. 2016;57(12):219.
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© 2017 Association for Research in Vision and Ophthalmology.
To create and characterize a tet-On transgenic Krt4-rtTA mouse line via CRISPR/Cas9 genome editing technique, which can modify gene expression and functions in mucosal epithelium, e.g., conjunctiva, oral mucosal epithelium.
A targeting construct of IRES-rtTA transgene flanked by Krt4 genomic DNA fragments was co-injected with a plasmid DNA of sgRNA and SpCas9 into the B6D2F2 mouse fertilized eggs that were subsequently implanted to pseudo-pregnant foster mothers. The new born pups were genotyped by PCR with specific Krt4 primers flanking the targeted region. The pups derived from homologous recombination event were identified and crossed with tet-O-histone2-EGFP (TH2) reporter mice. The expression of green fluorescent Histone-EGFP fusion protein is induced by feeding doxycycline to a pregnant and nursing moms or to the weaned mice. The expression pattern of Histone-EGFP fusion protein was examined by fluorescence stereomicroscope. Experimental mice were sacrificed at different postnatal ages, cryo sections were examined by a ZEISS fluorescence microscope.
The expression of green fluorescent protein was detected in conjunctiva, oral mucosa, eyelids of postnatal pups and adults. Spotted green fluorescent signals were also detected in corneal periphery. Examination of Cryo sections reveals green fluorescence-positive cells in conjunctival epithelium and goblet cells and acinar cells of meibomian glands and hair follicles. Cessation of doxycycline quickly diminished the number of green fluorescence-positive cells in conjunctiva, suggesting a quick turn-over of conjunctival epithelial cells.
The expression of rtTA reporter faithfully recapitulates the expression pattern of Krt4 in ocular surface epithelium, e.g., conjunctiva, meibomian glands, etc.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.
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