中国生物工程杂志

Four engineered zinc finger nucleases were produced for cleaving the internal transcribed spacers(ITS) of the human rRNA gene family to generate double-strand breaks, thereby increasing the efficiency of gene targeting. This study will lay a firm foundation for further in vivo studies of gene thrapy with gene targeting. First, two 9 bp-long appropriate sequences (with an internal sequence of 6 bp) within the ITS1 of the human rRNA gene family were selected as the recognition loci of zinc finger proteins. Based on the sequences of the recognition loci, two tri-zinc finger proteins were designed for each locus. The full-length DNA encoding zinc finger proteins were obtained through the gene splicing by overlap extension PCR (SOE PCR) method, then they were inserted into the expression vector pET-28a(+) to generate recombinant plasmid pET28a-ZFP. The zinc finger fusion proteins carrying a His-tag were produced by transformation of the recombinant plasmids into bacteria RossettaTM（DE3）. At the same time, the coding sequences of four zinc finger proteins were linked with the sequences encoding the DNA-cutting domain of Fok I, respectively, by SOE PCR. The resulting sequences encoding four zinc finger nucleases were then inserted into the expression vector pET-28a(+) to construct recombinant plasmid pET28a-ZFN. By transformation the recombinant plasmids into bacteria RossettaTM（DE3）, four zinc finger nuclease fusion proteins carrying His-tag were produced and purified.