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.