Multi-locus, high efficiency gene targeting mediated by zinc finger nucleases

To address the technical problems of producing stable transgenic animals and clinical applications of gene therapy, the present study aimed to establish a multi-locus gene targeting technique mediated by zinc finger nucleases (ZFN) that enables efficient site-specific integration and stable expression of foreign genes. First, the OPEN method (oligomerized pool engineering) was used to design four pairs of genes of zinc finger proteins (ZFP) that identify the internal transcribed spacer (ITS) of human ribosomal DNA (rDNA) genes. To obtain ZFN genes, the ZFP genes were synthesized by PCR and connected to the DNA sequences of the cutting domain of the endonuclease, Fok I. These ZFN genes were then cloned into eukaryotic expression vectors. In addition, vectors for multi-locus gene targeting containing two homologous recombination direct sequences and the EGFP gene were constructed. The two types of vector were co-transfected into HEK293 cells and efficient homologous recombination was induced by the ZFN cleaving the target sites of ITS of the rDNA genes. Site-specific integration of foreign genes was detected by internal reference control PCR and the gray analysis method. The efficiency using only multi-locus gene targeting vectors was 6.8%, while it was 24.2% following co-transfection of both the eukaryotic ZFN expression vectors and the multi-locus gene targeting vectors. Compared to the efficiency of 10-6-10-5 for conventional gene targeting, the efficiency of site-specific integration of foreign genes has been greatly improved (increased by more than 24000 times). After co-transfected HEK293 cells were cultured for two months (20 generations) without any drug selection, cells continued to express GFP. This study indicates that the technique of multi-locus gene targeting mediated by ZFN can not only greatly improve the efficiency of gene targeting, but can also produce stable expression of transgenes. A new technology platform for producing site-specific transgenic animals and with significant potential for use in human gene therapy has been established.