Congenital and acquired corneal diseases are exemplified by altered genetic functions. To better treat corneal diseases, it is imperative to gain insights of gene functions during embryonic development and pathogenesis in adults. To determine the molecular and cellular mechanisms of corneal epithelial differentiation and wound healing, a mouse line expressing Cre recombinase by the corneal epithelium has been prepared by knock-in the phage Cre gene into the Krt12 allele via gene targeting techniques. Additional mouse lines using Cre/IoxP system are prepared from the Krt12Cre knock-in mice for corneal epithelium-specific expression of green fluorescent protein, conditional ablations of Jun and Fos in compound floxed transgenic mice, Krt12Cre/Cre-RosaSF, Krt12Cre/Cre-Junf/f (homozygous floxed Jun) and Krt12Cre/Cre-Fosf/f, respectively.
Aim 1 is to determine differentiation and wound healing of corneal epithelium:
Aim 1 a is to examine the hypothesis that one of the two Krt12 alleles is preferentially used by individual corneal epithelial cells, Aim 1b is to determine the kinetics of differentiation during embryonic development and post natal growth of cornea, Aim 1c tests the hypothesis that corneal TAC (transit amplifying cells) cells of young mice are stem cell-like and of stem cell characteristics. To examine roles of AP-1 transcription factors on homeostasis of corneal epithelium and healing of corneal epithelium debridement, conditional Jun and Fos knockout mice (Krt12Cre/Cre-Junf/f and Krt12Cre/Cre-Fosf/f) are prepared.
Aim 2 is to determine roles of Jun on cornea-type epithelium differentiation and wound healing. Corneas of adult Krt12Cre/Cre-Junf/f mice will be subject to immunohistochemistry, northern and in situ hybridization, to determine the roles of Jun on homeostasis of corneal epithelium. The AP-1 dimers will be determined by immune precipitation with anti-Fos antibodies and western blot with anti-Jun family member. The nuclear and cytosol proteins isolated from injured and un-injured Krt12Cre/Cre-Junf/f and Krt12+/+-Junf/f (control) corneas will be subject to proteomic analysis using 2-dimensional High Performance Liquid Electrophoresis (2D-HPLE).
Aim 3 is to examine roles of Fos on corneal-type epithelium differentiation and corneal wound healing as described in Aim 2.
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