Journal
GENETICS
Volume 195, Issue 3, Pages 703-+Publisher
GENETICS SOCIETY AMERICA
DOI: 10.1534/genetics.113.156570
Keywords
TALENs; disease model; one-cell embryo; mouse mutant; nuclease; Fus; C9ORF72
Categories
Funding
- European Union [LSHG-CT-2005-018931]
- German Ministry of Education and Research [01GS0858]
- National Genome Research Network (NGFN)-Plus program
- excellence cluster for systems neurology (SyNergy) [1010]
- Kompetenznetzwerk Degenerative Demenzen (KNDD2) [FKZ01GI1005D]
- Indian Council of Agricultural Research [29-1/2009-EQR/Edn]
- European Research Council under the European Union [321366-Amyloid]
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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.
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