Development of an Effecient System for Gene Targeting

靶向突变是分子医学中一种非常有用的实验方法，它允许产生对任何感兴趣的基因都是突变的特殊动物。目前，任何基因打靶实验的速率决定步骤都是构建打靶载体(TV)。为了简化基因打靶方法，避免使用质粒TV遇到的问题，我们描述了噬菌体在定向突变中的直接应用。重组熟练的噬菌体载体2TK允许通过传统的限制性连接或重组介导的方法产生TV。然后，得到的TV DNA可以被限制性内切酶切割，释放噬菌体手臂，然后可以直接电穿孔到ES细胞中，产生基因靶点。我们证明，在体内，噬菌体-质粒重组可以通过双交叉重组将neo和LacZ-neo突变引入2TK亚克隆的精确位置。我们描述了消除单交叉重组子的两种方法：SPI选择和大小限制；这两种方法都会导致噬菌体电视带有双交叉插入。因此，TVS可以在噬菌体中方便、快速地产生，而不需要质粒亚克隆，并且几乎没有基因组序列或限制性内切酶位点的信息。

Targeted mutagenesis is an extremely useful experimental approach in molecular medicine, allowing the generation of specialized animals that are mutant for any gene of interest. Currently, the rate determining step in any gene targeting experiment is the construction of the targeting vector (TV). In order to streamline gene targeting methods and avoid problems encountered with plasmid TVs, we describe the direct application of phage in targeted mutagenesis. The recombination-proficient phage vector, 2TK,permits the generation of TVsby conventional restrictin-ligation or recombination-mediated methods. The resulting TV DNA can then be cleaved with restriction endonucleases to release the bacteriophage arms and can subsequently be electroporated directly into ES cells to yield gene targets. We demonstrate that in vivo phage-plasmid recombination can be used to introduce neo and lacZ-neo mutations into precise positions within a 2TK subclone via double-crossover recombination. We describe two methods for eliminating single-crossover recombinants : spi selection and size restriction ; both which result in phage TVs bearing double-crossover insertions. Thus TVs can be easily and quickly generated in bacteriophage without plasmid subcloning and with little genomic sequence or restriction site information.