Highly Efficient Targeted Mutagenesis in Mice Using TALENs
Highly Efficient Targeted Mutagenesis in Mice Using TALENs
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DOI：
10.1534/genetics.113.156570
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发表时间：
2013-11-01
期刊：
影响因子：
3.3
通讯作者：
Kuehn, Ralf
中科院分区：
文献类型：
--
作者：
Panda, Sudeepta Kumar;Wefers, Benedikt;Kuehn, Ralf
靶向小鼠突变体有助于分析健康和疾病中的基因功能。我们最近提供了在单细胞胚胎中使用转录激活剂样效应核酸酶 (TALEN) 进行小鼠基因组快速诱变的原理验证。在此，我们报告了一种有效生产疾病相关敲入和敲除突变体的常规程序，使用改进的 TALEN mRNA，其中包括质粒编码的聚腺苷酸尾 (TALEN-95A)，从而规避了有问题的体外聚腺苷酸化步骤。为了敲除 C9orf72 基因作为额颞叶变性模型，TALEN-95A 诱变诱导了 41% 的来自显微注射胚胎的幼崽的序列缺失。使用 TALEN 与诱变寡脱氧核苷酸一起，我们在肉瘤融合 (Fus) 基因中引入了肌萎缩侧索硬化症患者来源的错义突变，发生率为 6.8%。为了简单鉴定 TALEN 诱导的突变体及其后代，我们验证了 PCR 产物的高分辨率熔解分析 (HRMA) 作为一种灵敏且通用的基因分型工具。此外，突变始祖小鼠脱靶位点的 HRMA 未发现任何证据表明 TALEN 介导的相关基因组序列加工。用于增强诱变的 TALEN-95A mRNA 与用于简化基因分型的 HRMA 相结合，能够加速常规生产用于遗传疾病机制研究的新小鼠模型。
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.