June 2017
Volume 58, Issue 8
Open Access
ARVO Annual Meeting Abstract  |   June 2017
The generation and characterization of a novel mouse line, Cdh2- rtTA (Cdh2rt), for genetic manipulation of stem cell niche tissues
Author Affiliations & Notes
  • Yujin Zhang
    Optometry, Indiana University School of Optometry, Bloomington, Indiana, United States
  • Lingling Zhang
    Optometry, Indiana University School of Optometry, Bloomington, Indiana, United States
  • Diego G Ogando
    Optometry, Indiana University School of Optometry, Bloomington, Indiana, United States
  • Yuka Okada
    Optometry, Indiana University School of Optometry, Bloomington, Indiana, United States
    Wakayama Medical University, Wakayama, Japan
  • Yen-Chiao Wang
    Optometry, Indiana University School of Optometry, Bloomington, Indiana, United States
  • Winston W Y Kao
    Ophthamology, university of Cincinnati, Cincinnati, Ohio, United States
  • Yueh-Chiang Hu
    Division of Developmental Biology, Cincinnati Children’s Hospital Medical Center, Cincinnti, Ohio, United States
  • Joseph A Bonanno
    Optometry, Indiana University School of Optometry, Bloomington, Indiana, United States
  • Chia-Yang Liu
    Optometry, Indiana University School of Optometry, Bloomington, Indiana, United States
  • Footnotes
    Commercial Relationships   Yujin Zhang, None; Lingling Zhang, None; Diego Ogando, None; Yuka Okada, None; Yen-Chiao Wang, None; Winston Kao, None; Yueh-Chiang Hu, None; Joseph Bonanno, None; Chia-Yang Liu, None
  • Footnotes
    Support  NIH grant RO1 EY 21501 and EY 23086, RO1 EY011845, R01 021768, Research to Prevent Blindness, Ohio Lions Eye Research Foundation
Investigative Ophthalmology & Visual Science June 2017, Vol.58, 3905. doi:
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      Yujin Zhang, Lingling Zhang, Diego G Ogando, Yuka Okada, Yen-Chiao Wang, Winston W Y Kao, Yueh-Chiang Hu, Joseph A Bonanno, Chia-Yang Liu; The generation and characterization of a novel mouse line, Cdh2- rtTA (Cdh2rt), for genetic manipulation of stem cell niche tissues. Invest. Ophthalmol. Vis. Sci. 2017;58(8):3905.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose : Cell adhesion molecule such as N-cadherin (Cdh2) that mediates stem cell-niche interactions is crucial for stem cell self-renewal and is dynamically regulated. To achieve specific genetic modifications in stem cell-niche interaction during embryonic development and adult homeostasis, a novel Cdh2-rtTA knock-in (Cdh2rt) mouse line was generated.

Methods : N-cadherin (Chd2) is expressed in multiple tissues including retina and corneal endothelium and functions to mediate cell-cell adhesion. A gene-targeting vector containing a self-cutting peptide (P2A)-reverse tetracycline transcription activator (P2A-rtTA) cassette was constructed and fused P2A-rtTA cassette to the stop codon of Cdh2 via CRISPR/Cas technique so that a novel knock-in mouse line was generated. The resulting Cdh2rt mouse line was bred with TetO-Hist1H2BJ/GFP47Efu/J (TH2-GFP) reporter mouse line to obtain Cdh2rt/TH2-GFP bi-transgenic mice in which cells expressing Cdh2 are labeled by green fluorescence of GFP by doxycycline (Dox) induction for different time points. The GFP fluorescence in Cdh2rt/TH2-GFP was examined under a Zeiss stereomicroscope. .

Results : One germ line founder of Cdh2rt was successfully obtained through CRISPR/Cas genome engineering technology. When Cdh2rt /TH2-GFP bi-transgenic mice were subjected to Dox induction from embryonic day 0 (E0) to postnatal day 1(P1) and P30 separately, strong EGFP fluorescence labeled cells were specifically detected in corneal limbal stroma region, leptomeninges of brain and spinal cord, photoreceptor cells of retina and bone marrow. Surprisingly, however, there is no EGFP fluorescence detected in corneal endothelium.

Conclusions : The novel Cdh2rt mice can drive expression of rtTA in corneal limbal stroma, photoreceptor cells and meningeal cells. Since both limbal stromal and meningeal cells have potential role as stem cell niche harboring endogenous precursors that can be activated by injury and are able to contribute to tissue parenchymal reaction. This mouse line will be a useful reagent for gene manipulating and elucidating the biological functions in limbal stromal niche cells, meningeal niche cell and retinal photoreceptor cells during embryonic development, maintenance of homeostasis and pathogenesis.

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

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