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Open Access Green möglich sobald Postprint bei der ZB eingereicht worden ist.
Highly efficient targeted mutagenesis in mice using TALENs.
Genetics 195, 703-713 (2013)
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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Publikationstyp
Artikel: Journalartikel
Dokumenttyp
Wissenschaftlicher Artikel
Schlagwörter
Talens ; Disease Model ; One-cell Embryo ; Mouse Mutant ; Nuclease ; Fus ; C9orf72; Zinc-finger Nucleases; Amyotrophic-lateral-sclerosis; Homologous Recombination; Embryo Microinjection; Hexanucleotide Repeat; Mammalian-cells; Gene; C9orf72; Genome; Mouse
ISSN (print) / ISBN
0016-6731
e-ISSN
0016-6731
Zeitschrift
Genetics
Quellenangaben
Band: 195,
Heft: 3,
Seiten: 703-713
Verlag
Genetics Society of America
Nichtpatentliteratur
Publikationen
Begutachtungsstatus
Peer reviewed
Institut(e)
Institute of Developmental Genetics (IDG)