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A resource of vectors and ES cells for targeted deletion of microRNAs in mice

Abstract

The 21–23 nucleotide, single-stranded RNAs classified as microRNAs (miRNA) perform fundamental roles in diverse cellular and developmental processes. In contrast to the situation for protein-coding genes, no public resource of miRNA mouse mutant alleles exists. Here we describe a collection of 428 miRNA targeting vectors covering 476 of the miRNA genes annotated in the miRBase registry. Using these vectors, we generated a library of highly germline-transmissible C57BL/6N mouse embryonic stem (ES) cell clones harboring targeted deletions for 392 miRNA genes. For most of these targeted clones, chimerism and germline transmission can be scored through a coat color marker. The targeted alleles have been designed to be adaptable research tools that can be efficiently altered by recombinase-mediated cassette exchange to create reporter, conditional and other allelic variants. This miRNA knockout (mirKO) resource can be searched electronically and is available from ES cell repositories for distribution to the scientific community.

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Figure 1: Schematic illustration of targeting vector construction.
Figure 2: Targeting and reporter modification of the mir-290295 cluster.
Figure 3: Targeting and reporter modification of the mir-21 gene.
Figure 4: Targeting and conditional modification of the mir-106a363 cluster.

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Acknowledgements

We thank Y. Mok, D. Thomas, M. Li and G. Duddy for assistance with sections of the project. For database support and high-throughput vector designs, we are indebted to V. Iyer, D. Oakley, W. Yang, D. Klose and S. Briois. We thank A. West for advice on long range PCR genotyping. We are grateful for the advice and reagents for high-throughput vector construction received from P. Liu. We thank E. Miska for helpful comments on the manuscript. We thank R. Rad for advice with Q-PCR. We thank the Sanger Institute mouse mutant generation and animal facilities teams. We thank the public distribution repositories at MMRRC University of California Davis, USA, and as part of the EUCOMMTOOLS initiative EuMMCR at the Helmholtz Zentrum München, Munich, Germany, for agreeing to support the future distribution of the mirKO resource. This work was funded by the Wellcome Trust (WT079643). The IKMC database was developed with funding from the European Union and the US National Institutes of Health.

Author information

Authors and Affiliations

Authors

Contributions

H.M.P. planned and managed the project, constructed novel plasmid reagents and performed high-throughput vector construction, ES cell targeting experiments, post-targeting modifications, collated data and wrote the paper. H.K.-Y. performed high-throughput vector construction, planned and performed the ES cell targeting experiments, ES cell genotyping and collated data. J.D.C. performed high-throughput vector construction, ES cell targeting and genotyping. F.C.L. performed ES cell targeting experiments. A.B. initiated and supervised the project and wrote the paper.

Corresponding author

Correspondence to Haydn M Prosser.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary Table 2 and Supplementary Figures 1–4 (PDF 5058 kb)

Supplementary Table 1

Current status of individual mirKO targeting projects. (XLS 141 kb)

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Prosser, H., Koike-Yusa, H., Cooper, J. et al. A resource of vectors and ES cells for targeted deletion of microRNAs in mice. Nat Biotechnol 29, 840–845 (2011). https://doi.org/10.1038/nbt.1929

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