Preparation of K12-Cre Knock-In Mice by Gene Targeting Techniques

Abstract

Abstract: : Purpose: To better understand corneal epithelium cell biology, we have created a knock-in mouse line that expresses Cre recombinase in corneal epithelium, which can be used to ablate floxed genes in a cornea epithelium specific manner. Methods: A targeting construct containing intron 2 to exon 8 of Krt1.12 gene was prepared in which an IRES-Cre and the pgk-Neo reporter genes were inserted right after the stop codon within exon 8. Germ line chimera mice were obtained via conventional gene targeting techniques using embryonic stem cells. The K12-Cre mice were crossed with reporter ZEG mice that harbor a transgene containing a chicken ß actin promoter, a floxed lacZ gene (two loxP elements flanked at 5’and 3’ ends of lacZ) and EGFP in tandem. The Cre activity was assessed by the detection of green fluorescence in corneas of the offspring. Results: Seven clones of homologous recobinants were isolated and identified from 674 clones picked from electroporated ES cells. Homologous recombinant ES cells, clone K68, was used for blastocyst injection. One out of three chimeras was a germ line and yielded offspring carrying one allele of modified Krt1.12 gene. The K12-Cre knock-in mice were crossed with ZEG mice. Green fluorescence was detected only in compound transgenic K12 Cre+/-·ZEG mice. Conclusions: K12-Cre knock-in mice can be used to created mouse lines in which a any fuctional floxed genes will be ablated in corneal epithelium. Thus, it will allow us to investigate the role of a gene in corneal morphogenesis and homeostais via loss of function without threatening the life of the experimental animals.

Keywords: transgenics/knock-outs • cornea: epithelium • gene/expression