Division of Population Genetics, National Institute of Genetics, Mishima, Shizuoka, Japan.
Genomics. 2010 May;95(5):306-11. doi: 10.1016/j.ygeno.2010.02.006. Epub 2010 Feb 26.
Creating transgenic mice is an important technology for genetic studies and is currently performed by pronuclear microinjection of plasmid DNA into fertilized eggs. Since survival of injected embryos and integration of plasmid DNA are not efficient, total efficiency is only around 3% with a standard protocol. To circumvent this problem, here we describe a novel transgenesis method, the Tol2-mediated cytoplasmic injection method (Tol2:CI). We injected a foreign DNA cloned in a Tol2-transposon vector together with the transposase mRNA into the cytoplasm of fertilized eggs. As expected, the survival rate of the injected embryos was increased drastically. Also, the foreign DNA was transposed from the plasmid to the genome and transmitted to the next generation very efficiently. Together, the overall transgenic efficiency became more than 20%. Considering its simplicity and perfect compatibility with existing pronuclear microinjection facilities, we propose that the Tol2:CI method is applicable to high throughput functional genomics studies.
创建转基因小鼠是进行遗传研究的一项重要技术，目前主要通过将质粒 DNA 经原核显微注射到受精卵中实现。由于注射胚胎的存活率和质粒 DNA 的整合效率都不高，因此按照标准方案，总效率仅为 3%左右。为了解决这个问题，我们在这里描述了一种新的转基因方法，即 Tol2 介导的细胞质注射法（Tol2:CI）。我们将克隆在 Tol2 转座子载体中的外源 DNA 与转座酶 mRNA 一起注射到受精卵的细胞质中。正如预期的那样，注射胚胎的存活率大大提高。此外，外源 DNA 从质粒转座到基因组，并非常有效地传递到下一代。总体而言，转基因效率超过 20%。考虑到其简单性和与现有原核显微注射设备的完美兼容性，我们认为 Tol2:CI 方法适用于高通量功能基因组学研究。
