Rashmi Srivastava | Search PubMed

PubMed2026

Generation of miR-141/200c conditional knockout mice from knockout-first, reporter-tagged parent and functional validation of the floxed allele.

MicroRNAs (miRNAs) of the miR-200 family-specifically miR-141 and miR-200c-regulate neurogenesis, differentiation, and epithelial-mesenchymal transitions in development. Dysregulation of these miRNAs is associated with several diseases including cancer and stroke. The Mirc13tm1Mtm/Mmjax mouse line, which targets the miR-141/200c cluster, was originally generated and described by Park et al. 2012 as a knockout-first, reporter-tagged insertion with conditional potential (conditional-ready) mouse line. Harnessing its full potential requires a two-step breeding process: breeding with FLP mice to excise the lacZ/neo cassette, then breeding with Cre to delete the floxed miRNA cluster (Park et al. 2012). However, many studies either bypassed removal of the lacZ/Neo cassettes and treated the mouse line as Mirc13 knockouts or bred directly with Cre mouse lines, which could lead to unpredictable recombination and genotypes. Here we show that retention of the lacZ/Neo cassette is associated with reduced expression of the neighboring genes Ptpn6, Phb2 and Atn1 in the olfactory bulb, and that these genes are expressed normally once the cassette is excised. We therefore recommend a validated two-step FLPo-then-Cre breeding plan for this line, together with case-by-case allele validation for other knockout-first, reporter-tagged mouse lines.

Animals↗

PubMed2025

Validation and Optimization of Breeding Strategy for miR-141/200c Knockout Mice to Eliminate Off-Target Gene Silencing using FLPo Deleter.

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 Mirc13tm1Mtm /Mmjax mouse line, which targets the miR-141/200c cluster, was originally generated and 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 (1). However, subsequent studies either 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 or rare studies used less efficient FLPe Deleter mice. Here, we present a detailed and refined strategy to conditional miR-141/200c knockouts mice using FLPo Deleter mice to efficiently eliminate lacZ/neo cassettes. Our approach not only confirmed complete deletion of miR-141 and miR-200c in various organs such olfactory bulbs and lungs where these miRNAs are robustly expressed using various approach such as genotyping qPCR validation and in situ hybridization but showed that without the use of FLPo deleter mice deletion of miR-141/200c cluster amy also lead to loss of several close proximity physiologically important genes such as ptpn6, phb2, atn1 and eno1. By restoring a clean floxed allele using FLPo deleter mice 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.

Journal Article↗