Characterization of tet-On Abcb5 (P2A-rtTA) Driver Mice by CRISPR/Cas9-mediated Knock-in Strategy

Abstract

Purpose : To create and characterize an Abcb5-P2A-rtTA (Abcb5^R) knock-in mouse line that can modify gene functions in ocular surface epithelium and adnexa, e.g., conjunctiva, cornea & Meibomian gland

Methods : Abcb5^R was created by knocking-in a construct containing a P2A-rtTA transgene preceding the stop codon Abcb5 mRNA by CRSPR/Cas9. Thus, rtTA is produced, when Abcb5 is expressed in stem/progenitor cells of stratified epithelium and its adnexa, e.g., conjunctiva, limbus/cornea and Meibomian glands during doxycycline (Dox) induction. The knock-in mice are mated with reporter mice e.g., tet-O-histone2-EGFP (TH₂), tet-O-Cre(TC)/ROSA26^mTmG(mTmG) reporter mice to create double Abcb5^R/TH₂ and quadruple Abcb5^R/TC/TH2/mTmG mice. The expression of nuclear green fluorescent Histone-EGFP fusion protein and plasma membrane mG is induced by dox induction at different developmental and postnatal ages and chase the EGFP: label from E0-P0, P0-P21 and P21-P42 then chase for various periods of time from 0~8 weeks) by the cessation of Dox induction. The expression pattern of EGFP proteins was examined by fluorescence stereomicroscope and fluorescence microscopy

Results : The nuclear green fluorescence labels cells that express Abcb5 during Dox induction, when Dox is withdrawn, the nuclear green fluorescence gradually diminishes and disappears when cells continue to divide. In contrast, the non-dividing cells will remain nuclear fluorescence positive. Thus, one identifies the slow cycling (or label retaining) cells, a characteristic of stem/progenitor cells. Interestingly, when cells that are labeled by mG, they and their progeny will remain mG⁺ regardless of expression of Absb5 and Dox induction. Thereby, the fate of stem/progenitor cells can be traced. At the end of Dox induction, nuclear positive cells are found in conjunctival, limbal, peripheral cornea and Meibomian gland. At the cessation of Dox induction, the number of nuclear EGFP⁺ cells quickly diminished in cornea faster than conjunctiva and limbus. The number of mG positive cells are seen in conjunctiva, limbus and cornea epithelium. Cessation of Dox induction only slightly decrease in mG⁺ cells

Conclusions : The expression of rtTA reporter faithfully recapitulates the expression pattern of Abcb5 in ocular surface epithelium, e.g., conjunctiva, Corneal, Meibomian glands, etc.

This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.