title: Highly efficient targeted mutagenesis in mice using TALENs.
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.
authors: Institute of Developmental Genetics, Helmholtz Zentrum München, German Research Center for Environmental Health, 85764 Munich, Germany.
authors: Sudeepta Kumar
authors: SK
authors: Panda
authors: Benedikt
authors: B
authors: Wefers
authors: Oskar
authors: O
authors: Ortiz
authors: Thomas
authors: T
authors: Floss
authors: Bettina
authors: B
authors: Schmid
authors: Christian
authors: C
authors: Haass
authors: Wolfgang
authors: W
authors: Wurst
authors: Ralf
authors: R
authors: Kühn
mesh_terms: D000595:Amino Acid Sequence; D000690:Amyotrophic Lateral Sclerosis / Q000235:genetics; D000818:Animals; D001483:Base Sequence; D003851:Deoxyribonucleases / Q000235:genetics / Q000378:metabolism; D004195:Disease Models, Animal; D005260:Female; D055786:Gene Knockout Techniques / Q000379:methods; D005818:Genetic Engineering / Q000379:methods*; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout / Q000235:genetics; D008817:Mice, Mutant Strains / Q000235:genetics; D008822:Mice, Transgenic / Q000235:genetics; D008969:Molecular Sequence Data; D016296:Mutagenesis; D011247:Pregnancy; D012333:RNA, Messenger / Q000235:genetics; D034702:RNA-Binding Protein FUS / Q000235:genetics; D015533:Transcriptional Activation
chemical_list: D012333:RNA, Messenger; D034702:RNA-Binding Protein FUS; D003851:Deoxyribonucleases
keywords: C9ORF72; Fus; TALENs; disease model; mouse mutant; nuclease; one-cell embryo
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