Spatiotemporal gene targeting in the mouse corneal endothelium.

Abstract

The inducible Cre-ERT2 recombinase system allows for temporal control of gene targeting, and it is useful to studying adult function of genes that have critical developmental roles. The Zeb1flox/flox: UBC-CreERT2 mouse was generated to conditionally target Zeb1 to investigate its role in mesenchymal transition in the mouse corneal endothelium in vivo. Hemizygous UBC-CreERT2 mice were crossed with homozygous mice harboring loxP-flanked Zeb1 alleles (Zeb1flox/flox) to generate the Zeb1flox/flox: UBC-CreERT2 mouse. 4-hydroxytamoxifen (4-OHT) exposure leads to excision of exon 6 of Zeb1, resulting in a loss function allele in the Zeb1flox/flox: UBC-CreERT2 mouse. Intracameral 4-OHT injection further isolates Zeb1 targeting to the anterior chamber. Mesenchymal transition and induction of Zeb1 expression in the corneal endothelium was achieved using FGF2 in ex vivo organ culture. Gene expression was analyzed by semi-quantitative reverse transcription-polymerase chain reaction and by immunoblotting in the mouse corneal endothelium in vivo. Following Cre-mediated targeting of Zeb1 by intracameral 4-OHT injection in Zeb1flox/flox: UBC-CreERT2 mice, FGF2 treatment in ex vivo organ culture resulted in abrogation of Zeb1 mRNA and protein expression in the corneal endothelium. The data show Zeb1, a critical mediator of fibrosis in corneal endothelial mesenchymal transition, can be targeted by intracameral injection of 4-OHT in the mouse corneal endothelium in vivo. These results suggest that genes with critical developmental roles can be targeted at a specific time in the corneal endothelium to study its role in adult disease using an inducible Cre-Lox strategy.