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Generation of targeted mouse mutants by embryo microinjection of TALEN mRNA

Nature Protocols volume 8, pages 2355–2379 (2013)Cite this article

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Abstract

Genetically engineered mice are instrumental for the analysis of mammalian gene function in health and disease. As classical gene targeting, which is performed in embryonic stem (ES) cell cultures and generates chimeric mice, is a time-consuming and labor-intensive procedure, we recently used transcription activator–like (TAL) effector nucleases (TALENs) for mutagenesis of the mouse genome directly in one-cell embryos. Here we describe a stepwise protocol for the generation of knock-in and knockout mice, including the selection of TALEN-binding sites, the design and construction of TALEN coding regions and of mutagenic oligodeoxynucleotides (ODNs) and targeting vectors, mRNA production, embryo microinjection and the identification of modified alleles in founder mutants and their progeny. After a setup time of 2–3 weeks of hands-on work for TALEN construction, investigators can obtain first founder mutants for genes of choice within 7 weeks after embryo microinjections.

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Figure 1: Gene targeting in one-cell embryos using TALENs.
Figure 2: Design of TALEN target sites.
Figure 3: Construction of TALEN expression vectors.
Figure 4: Timeline of in silico and molecular work.
Figure 5: TALEN reporter plasmids and activity assay.
Figure 6: Flowchart and timing of mutant production.

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Acknowledgements

This work was supported by the European Union within the EUCOMMTools project (HEALTH-F4-2010-261492 to W.W.), by the German Ministry of Education and Research within the DIGTOP project (01GS0858 to W.W. and R.K.) of the German National Genome Research Network (NGFN)-Plus program and by the Indian Council of Agricultural Research (no.29-1/2009-EQR/Edn to S.K.P.).

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Authors and Affiliations

  1. Institute of Developmental Genetics, Helmholtz Zentrum München, German Research Center for Environmental Health, Munich, Germany

    Benedikt Wefers, Sudeepta K Panda, Oskar Ortiz, Christina Brandl, Svenja Hensler, Jens Hansen, Wolfgang Wurst & Ralf Kühn

  2. Technische Universität München, Freising-Weihenstephan, Germany

    Sudeepta K Panda, Christina Brandl, Svenja Hensler, Wolfgang Wurst & Ralf Kühn

  3. Deutsches Zentrum für Neurodegenerative Erkrankungen (DZNE), Munich, Germany

    Wolfgang Wurst

  4. Max Planck Institute of Psychiatry, Munich, Germany

    Wolfgang Wurst

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  1. Benedikt Wefers
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Contributions

B.W., S.K.P., O.O., C.B. and R.K. performed the research and analyzed the data; B.W., S.K.P., O.O., C.B., S.H. and R.K. wrote the manuscript; J.H. designed the TALENdesigner tools and webpage; W.W. and R.K. supervised the research.

Corresponding author

Correspondence to Ralf Kühn.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Genotyping: exemplary results for direct sequencing of PCR products.

Representative chromatograms of heterozygous mutants harboring a single nucleotide substitution (marked in red) (a) or an indel (b) resulting in two superimposed traces.

Supplementary Figure 2 Genotyping: exemplary results of a T7 endonuclease I assay.

Wild-type controls (wt) harbor only the full length PCR product (open triangle), whereas heterozygous mutants (het) show the additional presence of two digestion products (filled triangles) obtained by the cleavage of heteroduplex molecules within the TALEN target region.

Supplementary Figure 3 Genotyping: exemplary results for HRMA analysis.

Representative melting curves of PCR products amplified from a wild-type control (grey) and a mutant founder mouse (red).

Supplementary information

Supplementary Figure 1

Genotyping: exemplary results for direct sequencing of PCR products. (PDF 155 kb)

Supplementary Figure 2

Genotyping: exemplary results of a T7 endonuclease I assay. (PDF 148 kb)

Supplementary Figure 3

Genotyping: exemplary results for HRMA analysis. (PDF 27 kb)

Supplementary Data

(PDF 889 kb)

Pronuclear microinjection

This movie demonstrates the microinjection into the male pronucleus of a mouse one-cell embryo, fixed with a holding pipette. (AVI 2868 kb)

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Wefers, B., Panda, S., Ortiz, O. et al. Generation of targeted mouse mutants by embryo microinjection of TALEN mRNA. Nat Protoc 8, 2355–2379 (2013). https://doi.org/10.1038/nprot.2013.142

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