Generation and characterization of a novel mouse line, keratocan-rtTA (KeraRT), for corneal stroma and tendon research

Yujin Zhang, Winston W.Y. Kao, Yasuhito Hayashi, Lingling Zhang, Mindy Call, Fei Dong, Yong Yuan, Jianhua Zhang, Yen Chiao Wang, Okada Yuka, Atsushi Shiraishi, Chia Yang Liu

Research output: Contribution to journalArticlepeer-review

8 Scopus citations

Abstract

PURPOSE. We created a novel inducible mouse line Keratocan-rtTA (KeraRT) that allows specific genetic modification in corneal keratocytes and tenocytes during development and in adults. METHODS. A gene-targeting vector (Kera-IRES2-rtTA3) was constructed and inserted right after the termination codon of the mouse Kera allele via gene targeting techniques. The resulting KeraRT mouse was crossed to tet-O-Hist1H2B-EGFP (TH2B-EGFP) to obtain KeraRT/TH2B-EGFP compound transgenic mice, in which cells expressing Kera are labeled with green fluorescence protein (GFP) by doxycycline (Dox) induction. The expression patterns of GFP and endogenous Kera were examined in KeraRT/TH2B-EGFP. Moreover, KeraRT was bred with tet-O-TGF-α to generate a double transgenic mouse, KeraRT/tet-O-TGF-α, to overexpress TGF-a in corneal keratocytes upon Dox induction. RESULTS. Strong GFP-labeled cells were detected in corneal stroma, limbs, and tail when KeraRT/TH2B-EGFP mice were fed Dox chow. There was no GFP in any single transgenic KeraRT or TH2B-EGFP mouse. Histological analysis showed that GFP in the cornea was limited to stromal keratocytes of KeraRT/TH2B-EGFP, which is consistent with Kera expression. Induction of GFP occurred in 24 hours and reached a plateau by 7 days after Dox induction. GFP could be detected 3-months after induction of KeraRT/TH2B-EGFP. Ectopic expression of TGF-α in corneal keratocytes caused hyperplasia in the corneal epithelium and stroma. CONCLUSIONS. The novel Dox inducible KeraRT driver mouse line is a useful genetic tool for gene manipulation and elucidating gene functions in corneal stroma and tendons of limbs and tail during embryonic development, homeostasis and pathogenesis.

Original languageEnglish (US)
Pages (from-to)4800-4808
Number of pages9
JournalInvestigative Ophthalmology and Visual Science
Volume58
Issue number11
DOIs
StatePublished - Sep 2017
Externally publishedYes

Bibliographical note

Funding Information:
Supported by grants from the National Institutes of Health/ National Eye Institute (NIH/NEI, Bethesda, MD, USA) R01 EY21501, EY23086 (to CYL), EY011845 (to WWYK), Research Prevent Blindness and Ohio Lions Foundation for Eye Research. Disclosure: Y. Zhang, None; W.W.-Y. Kao, None; Y. Hayashi, None; L. Zhang, None; M. Call, None; F. Dong, None; Y. Yuan, None; J. Zhang, None; Y.-C. Wang, None; O. Yuka, None; A. Shiraishi, None; C.-Y. Liu, None

Publisher Copyright:
© 2017 The Authors.

Keywords

  • Corneal stroma
  • Dox-inducible
  • GFP
  • Keratocan
  • Knock-in mouse model

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