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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. Access through your institution Buy or subscribe This is a preview of subscription content, access via your institution ACCESS OPTIONS

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PRODUCING GENETICALLY MODIFIED MICE Article Open access 03 June 2021 ELECTROPORATION AND GENETIC SUPPLY OF CAS9 INCREASE THE GENERATION EFFICIENCY OF CRISPR/CAS9 KNOCK-IN ALLELES IN C57BL/6J

MOUSE ZYGOTES Article Open access 21 October 2020 PRECISE ALLELE-SPECIFIC GENOME EDITING BY SPATIOTEMPORAL CONTROL OF CRISPR-CAS9 VIA PRONUCLEAR TRANSPLANTATION Article Open access 14

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effector genes. _Nat. Biotechnol._ 31, 76–81 (2013). Article  CAS  Google Scholar  Download references 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.). AUTHOR INFORMATION AUTHORS AND AFFILIATIONS * 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 * Technische Universität München, Freising-Weihenstephan, Germany Sudeepta K Panda, Christina Brandl, Svenja Hensler, Wolfgang Wurst & Ralf Kühn *

Deutsches Zentrum für Neurodegenerative Erkrankungen (DZNE), Munich, Germany Wolfgang Wurst * Max Planck Institute of Psychiatry, Munich, Germany Wolfgang Wurst Authors * Benedikt Wefers

View author publications You can also search for this author inPubMed Google Scholar * Sudeepta K Panda View author publications You can also search for this author inPubMed Google Scholar *

Oskar Ortiz View author publications You can also search for this author inPubMed Google Scholar * Christina Brandl View author publications You can also search for this author inPubMed 

Google Scholar * Svenja Hensler View author publications You can also search for this author inPubMed Google Scholar * Jens Hansen View author publications You can also search for this

author inPubMed Google Scholar * Wolfgang Wurst View author publications You can also search for this author inPubMed Google Scholar * Ralf Kühn View author publications You can also search

for this author inPubMed Google Scholar 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 TALEN_designer_ tools and webpage; W.W. and R.K. supervised the research. CORRESPONDING AUTHOR Correspondence to Ralf Kühn. ETHICS DECLARATIONS COMPETING

INTERESTS 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) RIGHTS AND PERMISSIONS Reprints and permissions ABOUT THIS

ARTICLE CITE THIS ARTICLE 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 Download citation * Published: 31 October 2013 * Issue Date: December 2013 * DOI: https://doi.org/10.1038/nprot.2013.142 SHARE THIS ARTICLE Anyone you

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