Dataset Information

Generation and Characterization of a Novel Mouse Line, Keratocan-rtTA (KeraRT), for Corneal Stroma and Tendon Research.

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-? to generate a double transgenic mouse, KeraRT/tet-O-TGF-?, to overexpress TGF-? 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-? 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.

SUBMITTER: Zhang Y

PROVIDER: S-EPMC5624774 | biostudies-literature | 2017 Sep

REPOSITORIES: biostudies-literature

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Publications

Generation and Characterization of a Novel Mouse Line, Keratocan-rtTA (KeraRT), for Corneal Stroma and Tendon Research.

Zhang Yujin Y Kao Winston W-Y WW Hayashi Yasuhito Y Zhang Lingling L Call Mindy M Dong Fei F Yuan Yong Y Zhang Jianhua J Wang Yen-Chiao YC Yuka Okada O Shiraishi Atsushi A Liu Chia-Yang CY

Investigative ophthalmology & visual science 20170901 11

<h4>Purpose</h4>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.<h4>Methods</h4>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 ...[more]

PMID: 28973326

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Project description:BackgroundThe pluripotent state in embryonic stem (ES) cells is controlled by a core network of transcription factors that includes Nanog, Oct4 and Sox2. Nanog is required to reach pluripotency during somatic reprogramming and is the only core factor whose overexpression is able to oppose differentiation-promoting signals. Additionally, Nanog expression is known to fluctuate in ES cells, and different levels of Nanog seem to correlate with ES cells' ability to respond to differentiation promoting signals. Elucidating how dynamic Nanog levels are regulated in pluripotent cells and modulate their potential is therefore critical to develop a better understanding of the pluripotent state.Methodology/principal findingsWe describe the generation and validation of a mouse ES cell line with a novel Nanog reporter (Nd, from Nanog dynamics), containing a BAC transgene where the short-lived fluorescent protein VNP is placed under Nanog regulation. We show that Nanog and VNP have similar half-lives, and that Nd cells provide an accurate and measurable read-out for the dynamic levels of Nanog. Using this reporter, we could show that ES cells with low Nanog levels indeed have higher degree of priming to differentiation, when compared with high-Nanog cells. However, low-Nanog ES cells maintain high levels of Oct4 and Sox2 and can revert to a state of high-Nanog expression, indicating that they are still within the window of pluripotency. We further show that the observed changes in Nanog levels correlate with ES cell morphology and that Nanog dynamic expression is modulated by the cellular environment.Conclusions/significanceThe novel reporter ES cell line here described allows an accurate monitoring of Nanog's dynamic expression in the pluripotent state. This reporter will thus be a valuable tool to obtain quantitative measurements of global gene expression in pluripotent ES cells in different states, allowing a detailed molecular mapping of the pluripotency landscape.

Dataset Information

Generation and Characterization of a Novel Mouse Line, Keratocan-rtTA (KeraRT), for Corneal Stroma and Tendon Research.

Purpose

Methods

Results

Conclusions

Publications

Generation and Characterization of a Novel Mouse Line, Keratocan-rtTA (KeraRT), for Corneal Stroma and Tendon Research.

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