Protocol for establishing primary murine colonic fibroblast culture with induction of loxP/loxP deletion using transducible TAT-Cre recombinase

Conditional mutagenesis using site-specific recombinases is widely used in transgenic mouse models; however, its efficiency in vitro is limited by poor tamoxifen activity. Here, we present an optimized in vitro system for Cre-mediated loxP/loxP deletion in primary colonic fibroblasts. First, we describe steps for isolating, culturing, and monitoring murine colonic fibroblasts. Second, we detail procedures for inducing recombination using transducible TAT-Cre recombinase, enabling controlled gene activation or deletion in vitro . For complete details on the use and execution of this protocol, please refer to Chulkina et al. 1 • Instructions for isolation and culture of primary murine colonic fibroblasts • Steps for TAT-Cre-induced loxP/loxP recombination • Details on protocol optimization, limitations, and possible solutions • Guidance on analysis of controlled gene activation or deletion Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics. Conditional mutagenesis using site-specific recombinases is widely used in transgenic mouse models; however, its efficiency in vitro is limited by poor tamoxifen activity. Here, we present an optimized in vitro system for Cre-mediated loxP/loxP deletion in primary colonic fibroblasts. First, we describe steps for isolating, culturing, and monitoring murine colonic fibroblasts. Second, we detail procedures for inducing recombination using transducible TAT-Cre recombinase, enabling controlled gene activation or deletion in vitro .