A method for TAT-Cre recombinase-mediated floxed allele modification in ex vivo tissue slices

Abstract

ABSTRACT Precision-cut lung slices (PCLS) are used for a variety of applications. However, methods to manipulate genes in PCLS are currently limited. We developed a new method, TAT-Cre recombinase-mediated floxed allele modification in tissue slices (TReATS), to induce highly effective and temporally controlled gene deletion or activation in ex vivo PCLS. Treatment of PCLS from Rosa26-flox-stop-flox-EYFP mice with cell-permeant TAT-Cre recombinase induced ubiquitous EYFP protein expression, indicating successful Cre-mediated excision of the upstream loxP-flanked stop sequence. Quantitative real-time PCR confirmed induction of EYFP. We successfully replicated the TReATS method in PCLS from Vangl2flox/flox mice, leading to the deletion of loxP-flanked exon 4 of the Vangl2 gene. Cre-treated Vangl2flox/flox PCLS exhibited cytoskeletal abnormalities, a known phenotype caused by VANGL2 dysfunction. We report a new method that bypasses conventional Cre-Lox breeding, allowing rapid and highly effective gene manipulation in ex vivo tissue models.