RESUMEN
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-α 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.
Asunto(s)
Sustancia Propia/metabolismo , Técnicas de Sustitución del Gen , Ratones Transgénicos/genética , Proteoglicanos/genética , Tendones/metabolismo , Animales , Queratocitos de la Córnea/metabolismo , Modelos Animales de Enfermedad , Epitelio Corneal/metabolismo , Técnicas de Inactivación de Genes , Vectores Genéticos , Proteínas Fluorescentes Verdes/metabolismo , Ratones , Proteoglicanos/metabolismo , Factor de Crecimiento Transformador alfa/genética , Factor de Crecimiento Transformador alfa/metabolismoRESUMEN
This protocol is developed for primary cell culture of cornea stromal keratocytes isolated from neonatal mouse eyeballs. It provides an optimal condition to isolate stromal keratocytes which maintain high viability for cell culture.