Redefined Strategies to generate Conditional miR-141/200c miRNA cluster Knockout mice to eliminate confounding neo cassettes.

MicroRNAs (miRNAs) of the miR-200 family specifically miR-141 and miR-200c regulate neurogenesis, differentiation, and epithelial-mesenchymal transitions in development and several diseases including cancer and stroke. The STOCK Mirc13 /Mmjax mouse line, which targets the miR-141/200c cluster, was originally described by Park et al. (2012) as a conditional "knockout-first" allele requiring a two-step breeding strategy: Flp recombination to excise lacZ/neo cassettes followed by Cre recombination to delete the floxed miRNA cluster. However, subsequent studies frequently bypassed this step and reported knockouts based on direct crosses with Cre mouse lines, leaving residual lacZ/neo sequences that may silence upstream elements or introduce transcriptional artifacts. Here, we present a detailed and refined strategy to generate both global and tissue-specific miR-141/200c knockouts that adheres to the original design intent and eliminates confounding cassettes. Our approach confirmed robust baseline expression of miR-141 and miR-200c in various organs such olfactory bulbs and lungs where these miRNAs are robustly expressed, with near-complete loss of expression in knockout animals as validated by qPCR and in situ hybridization. By restoring a clean floxed allele prior to Cre deletion, we establish a reliable and interpretable mouse model for dissecting the roles of the miR-141/200c cluster miRNA in various disease models in these mice.