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Highly efficient targeted mutagenesis in mice using TALENs

Item Type:Article
Title:Highly efficient targeted mutagenesis in mice using TALENs
Creators Name:Panda, S.K., Wefers, B., Ortiz, O., Floss, T., Schmid, B., Haass, C., Wurst, W. and Kühn, R.
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:TALENs, Disease Model, One-Cell Embryo, Mouse Mutant, Nuclease, Fus, C9ORF72, Animals, Mice
Publisher:Genetics Society of America
Page Range:703-713
Date:November 2013
Official Publication:https://doi.org/10.1534/genetics.113.156570
PubMed:View item in PubMed

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