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Jianhua Zhang, Yujin Zhang, Mindy Call, Chia-Yang Liu, Ruth Ashery-Padan, Winston W Y Kao; Generation and Characterization of Pax6-rtTA (P6R) Transgenic Mice for Genetic Manipulation of Ocular Surface Epithelium. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):5615.
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© ARVO (1962-2015); The Authors (2016-present)
The purpose of the present study is to generate rtTA2S-M2-VP16 driver mice for expressing tet-O-transgenes and/or deletion of floxed genes is achieved in the ocular surface epithelium via doxycycline (Dox) induction.
A 5’ 6440 bp genomic DNA fragment of the mouse Pax6 promoter (anterior segment-specific containing the ectoderm enhancer and P0 promoter) was ligated to rtTA2S-M2-VP16 cDNA and used to create transgenic mice via transgenesis. Fluorescent in situ hybridization (FISH) was employed to determine the chromosome location of the transgene. Bitransgenic P6R/TH2-GFP mice were obtained by breeding P6R and reporter mouse Tg(tetO-HIST1H2BJ/GFP)47Efu/J (TH2/GFP). Triple transgenic P6R/TC (tet-O-Cre)/mTmG (dual Cre reporter, (Gt(ROSA)26Sortm4(ACTB-tdTomato,-EGFP)Luo/J). The expression of histoneH2-GFP and ROSA26EGFP in bitransgenic and triple transgenic mice was induced by Dox chow to the pregnant dam (embryonic) and weaned pups (postnatal). The expression pattern of GFP was determined by fluorescence stereo microscopy using a fluorescence microscope (ZEISS).
Two independent transgenic founder lines were obtained. FISH analysis revealed that one transgene was inserted on the X-chromosome and is termed P6RX and the other is located on chromosome 11 termed P6R11. The expression pattern of HistoneH2GFP and ROSA26-EGFP are similar between these two lines. Embryonic induction resulted in expression of GFP in the epithelia of the cornea, conjunctiva, and lens. It was also found in the inner nuclear layer (INL) and ganglion layers (GL) of the retina. In contrast, postnatal induction showed GFP expression in the epithelia of cornea and conjunctiva, but not in the lens epithelium and retina. It is possible that the Dox may not reach the lens at postnatal stages as the hyaloid vessel supplying nutrient to the lens has already regressed, while the differentiated retinal cells in INL and GL may not express the Pax6-rtTA transgene, effectively.
The two transgenic mouse lines share similar expression patterns of GFP, suggesting that the DNA elements of this Pax6 promoter are sufficient for cornea and conjunctiva epithelium expression of the rtTA transgene. Thus, the two transgenic mouse lines can be used to manipulate gene expression in ocular surface epithelia.
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