| 摘要 |
Provided are a novel two-step gene targeting method and a nucleotide construct for gene targeting. The method can improve the gene targeting efficiency and accurately identify a target gene knock-out mutant. The method of the present invention comprises: firstly, efficiently replacing a target gene in a genome with a targeting box by homologous recombination, the targeting box consisting of a target gene activity variant, a marker gene and site-specific recombination sites; and secondly, resecting the targeting box by recombinase, leaving a site-specific recombination site on the target gene to generate a target gene knock-out mutant, and removing a recombinase expression vector from the knock-out mutant by using a counter selection marker in the recombinase expression vector. |
