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Genetics. 2013 Nov;195(3):703-13. doi: 10.1534/genetics.113.156570. Epub 2013 Aug 26.

Highly efficient targeted mutagenesis in mice using TALENs.

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  • 1Institute of Developmental Genetics, Helmholtz Zentrum München, German Research Center for Environmental Health, 85764 Munich, Germany.

Abstract

Targeted mouse mutants are instrumental for the analysis of gene function in health and disease. We recently provided proof-of-principle for the fast-track mutagenesis of the mouse genome, using transcription activator-like effector nucleases (TALENs) in one-cell embryos. Here we report a routine procedure for the efficient production of disease-related knockin and knockout mutants, using improved TALEN mRNAs that include a plasmid-coded poly(A) tail (TALEN-95A), circumventing the problematic in vitro polyadenylation step. To knock out the C9orf72 gene as a model of frontotemporal lobar degeneration, TALEN-95A mutagenesis induced sequence deletions in 41% of pups derived from microinjected embryos. Using TALENs together with mutagenic oligodeoxynucleotides, we introduced amyotrophic lateral sclerosis patient-derived missense mutations in the fused in sarcoma (Fus) gene at a rate of 6.8%. For the simple identification of TALEN-induced mutants and their progeny we validate high-resolution melt analysis (HRMA) of PCR products as a sensitive and universal genotyping tool. Furthermore, HRMA of off-target sites in mutant founder mice revealed no evidence for undesired TALEN-mediated processing of related genomic sequences. The combination of TALEN-95A mRNAs for enhanced mutagenesis and of HRMA for simplified genotyping enables the accelerated, routine production of new mouse models for the study of genetic disease mechanisms.

KEYWORDS:

C9ORF72; Fus; TALENs; disease model; mouse mutant; nuclease; one-cell embryo

PMID:
23979585
[PubMed - indexed for MEDLINE]
PMCID:
PMC3813858
Free PMC Article
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