Ludwig-Maximilians-Universität, Chair of Metabolic Biochemistry
print


Breadcrumb Navigation


Content

Highly Efficient Targeted Mutagenesis in Mice Using TALENs

Genetics. 2013 Aug 26. [Epub ahead of print]

Authors/Editors: Panda SK
Wefers B
Ortiz O
Floss T
Schmid B
Haass C
Wurst W
Kühn R
Publication Date: 2013
Type of Publication: Journal Article

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 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 knockout 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

Related Links