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H. Yi, E. Carlson, J. Ouyang, V. Perez, Y.–F. Lee, W.–Y. Kao, C.–Y. Liu; In vivo Identification of the Cis–Regulatory Elements of the Murine Keratocan (mKera) Gene . Invest. Ophthalmol. Vis. Sci. 2005;46(13):2608.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: To identify the elements of the mKerapr3.2–bpA promoter responsible for cornea–specific gene expression using deletion mutant analysis in vivo. Methods: The entire intron 1 sequence (1.5 kb) of mKera was inserted into mKerapr3.2–EGFP–bpA to generate mKerapr3.2–int–EGFP–bpA. A series of deletion mutant mKera promoters (–2.8kb, –2.0 kb, –1.1 kb, –0.8 kb, and –0.3 kb) or internal deletion of the intron 1 (0.5 kb) were generated by restriction enzyme digestion and re–ligation in the mKerapr3.2–int–EGFP–bpA vector. A 0.5 kb fragment of the intron 1 was inserted into the 3’ to the bpA of the mKerapr3.2–EGFP–bpA to generated mKerapr3.2–EGFP–bpA–int. Deletion mKera promoters and control plasmids were delivered in vivo to murine corneal stroma and conjunctiva by intrastromal and conjunctival injections. In vivo expression was determined by measuring EGFP using a stereomicroscope at different time points. After 24 hours eye were enucleated and analyzed further using fluorescent stereo and confocal microscopies. Results: In vivo transfection of corneas with the mKerapr3.2–int–EGFP–bpA promoter resulted in stronger in vivo expression. In contrast, the mKerapr3.2–EGFP–bpA construct resulted in poor EGFP reporter gene expression in corneal stroma cells. Interestingly, the mKerapr3.2–EGFP–bpA–int showed compatible EGFP expression to that of the mKerapr3.2–int–EGFP–bpA, indicating that the 0.5 kb intron 1 fragment contains a putative enhancer. In vivo deletion mutant analysis showed that deletion from –3.2 kb to –2.8 kb reduced promoter activity by ∼60% and further deletion to –1.1 kb resulted in the loss of promoter activity. However, deletions to –0.8 kb and –0.3 kb restored the promoter activity back to 40 % and 90 %, respectively, as compared to that of the –3.2 kb promoter. All of the mKera promoters tested showed cornea–specific EGFP expression Conclusions: These results show the ability of in vivo promoter analysis to identify cis–regulatory elements in mKera between –1.1 and –0.8 kb which serve as silencers that can be overcome by enhancers between –3.2 kb to –1.1 kb. Furthermore, we have identified mKera intron 1 as being crucial for its promoter activity in corneal stromal cells and the –0.3 kb mKera promoter region being sufficient to drive cornea–specific expression.
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