Disclosed herein are methods for generating recombinant DNA molecules in cells using homologous recombination mediated by recombinases and similar proteins. The methods promote high efficiency homologous recombination in bacterial cells, and in eukaryotic cells such as mammalian cells. The methods are useful for cloning, the generation of transgenic and knockout animals, and gene replacement. The methods are also useful for subcloning large DNA fragments without the need for restriction enzymes. The methods are also useful for repairing single or multiple base mutations to wild type or creating specific mutations in the genome. Also disclosed are bacterial strains and vectors which are useful for high-efficiency homologous recombination.
本文公开了利用
重组酶和类似蛋白介导的同源
重组在细胞中产生
重组 DNA 分子的方法。这些方法可促进细菌细胞和真核细胞(如哺乳动物细胞)中的高效同源
重组。这些方法可用于克隆、产生转
基因动物和
基因敲除动物以及
基因置换。这些方法还可用于亚克隆大的 DNA 片段,而无需使用限制性酶。这些方法还可用于将单个或多个碱基突变修复为野生型或在
基因组中产生特定突变。还公开了可用于高效同源
重组的细菌菌株和载体。