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Validation and Optimization of Breeding Strategy for miR-141/200c Knockout Mice to Eliminate Off-Target Gene
Rashmi Srivastava1, Sanjeev Kumar Yadav1, Mary-Catherine Cormier1
1Department of Neurosciences, UConn Health Farmington CT 06030.
Abstract:
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.
Insights
We refined a mouse model strategy for conditional miR-141/200c knockout, ensuring accurate gene deletion and avoiding artifacts. This improves research into microRNAs
Area of Science:
- Developmental Biology
- Molecular Biology
- Genetics
Background:
- MicroRNAs (miRNAs) of the miR-200 family, including miR-141 and miR-200c, are crucial regulators of neurogenesis, differentiation, and epithelial-mesenchymal transitions.
- These miRNAs play significant roles in development and diseases such as cancer and stroke.
- Existing conditional knockout mouse models for the miR-141/200c cluster present challenges due to incomplete cassette removal, potentially causing transcriptional artifacts.
Purpose of the Study:
- To develop a refined and reliable strategy for generating conditional knockout mice targeting the miR-141/200c miRNA cluster.
- To ensure efficient and complete deletion of the miR-141/200c cluster using FLPo deleter mice.
- To establish an interpretable mouse model for studying the roles of miR-141/200c in various biological processes and disease models.
Main Methods:
- Utilized a two-step breeding strategy involving FLPo recombination to efficiently excise lacZ/neo cassettes.
- Employed Cre recombination to delete the floxed miR-141/200c miRNA cluster.
- Validated complete miRNA deletion through genotyping, qPCR, and in situ hybridization in organs like olfactory bulbs and lungs.
Main Results:
- Successfully generated conditional miR-141/200c knockout mice with efficient lacZ/neo cassette elimination using FLPo deleter mice.
- Confirmed complete deletion of miR-141 and miR-200c in relevant tissues.
- Demonstrated that incomplete deletion without FLPo can lead to the unintended loss of adjacent genes (ptpn6, phb2, atn1, eno1).
Conclusions:
- The presented strategy provides a reliable method for creating clean conditional knockouts of the miR-141/200c cluster.
- This refined approach avoids artifacts associated with residual sequences and loss of neighboring genes.
- The established mouse model is suitable for accurate investigation of miR-141/200c functions in physiological and pathological contexts.
