Development of an Effecient System for Gene Targeting

靶向诱变在分子医学中是一种非常有用的实验方法，允许产生任何感兴趣的基因突变的特殊动物。目前，在任何基因靶向实验中，速率的决定步骤是靶向载体（TV）的构建。为了简化基因靶向方法，避免质粒电视遇到的问题，我们描述了噬菌体在靶向诱变中的直接应用。重组精通噬菌体载体，2TK，允许通过传统的限制性连接或重组介导的方法产生tvs。由此产生的TV DNA可以用限制性内切酶裂解以释放噬菌体臂，随后可以直接电穿孔到胚胎干细胞中以产生基因靶标。我们证明体内噬菌体-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。我们描述了两种消除单交叉重组的方法：spi选择和大小限制；两者都会导致噬菌体电视携带双重交叉插入。因此，无需质粒亚克隆，无需基因组序列或限制性位点信息，可以在噬菌体中轻松快速地生成TVs。

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.