Department of Systems BioMedicine, National Research Institute for Child Health and Development, Tokyo, Japan.
PLoS One. 2013 Oct 16;8(10):e76004. doi: 10.1371/journal.pone.0076004. eCollection 2013.
Mice are among the most valuable model animal species with an enormous amount of heritage in genetic modification studies. However, targeting genes in mice is sometimes difficult, especially for small genes, such as microRNAs (miRNAs) and targeting genes in repeat sequences. Here we optimized the application of TALEN system for mice and successfully obtained gene targeting technique in mice for intergenic region and series of microRNAs. Microinjection of synthesized RNA of TALEN targeting each gene in one cell stage of embryo was carried out and injected oocytes were transferred into pseudopregnant ICR female mice, producing a high success rate of the targeted deletion of miRNA genes. In our condition, TALEN RNA without poly(A) tail worked better than that of with poly(A) tail. This mutated allele in miRNA was transmitted to the next generation, suggesting the successful germ line transmission of this targeting method. Consistent with our notion of miRNAs maturation mechanism, in homozygous mutant mice of miR-10a, the non- mutated strand of miRNAs expression was completely diminished. This method will lead us to expand and accelerate our genetic research using mice in a high throughput way.
小鼠是最有价值的模式动物物种之一，在遗传修饰研究方面拥有大量的遗传资源。然而，在小鼠中靶向基因有时很困难，特别是对于小基因，如 microRNAs（miRNAs）和靶向重复序列中的基因。在这里，我们优化了 TALEN 系统在小鼠中的应用，并成功地获得了针对基因间区和一系列 microRNAs 的小鼠基因靶向技术。在胚胎的一个细胞阶段，将针对每个基因的 TALEN 靶向 RNA 进行微注射，并将注射的卵母细胞转移到假孕 ICR 雌性小鼠中，从而产生 miRNA 基因靶向缺失的高成功率。在我们的条件下，没有 poly(A) 尾的 TALEN RNA 比带有 poly(A) 尾的 RNA 效果更好。这种 miRNA 的突变等位基因被传递到下一代，表明这种靶向方法的成功的种系传递。与我们 miRNA 成熟机制的观点一致，在 miR-10a 的纯合突变小鼠中，miRNAs 的非突变链的表达完全消失。这种方法将使我们能够以高通量的方式扩展和加速使用小鼠的遗传研究。
