Zinc finger nucleases (ZFN), which are artificial restriction enzymes consisting of an engineered zinc-finger domain (ZF) and an endonuclease domain, can be used for the induction of site-directed mutation and the efficient generation of gene knockout animals. However, the repeated construction of various ZFN sequences is both expensive and time consuming. In this study, we attempted to establish a novel method for inexpensive and rapid ZFN construction. First, we constructed ZFN against mouse Rosa26 and original mouse Gli3 gene loci using short PCR primer sets (>30 bp), which contained 21 bp of the ZF recognition helix for a specific DNA triplet. We prepared 18 sets of such primers and PCR was performed using one of these primer sets and the partial ZF sequence as a template, which was obtained from the first to second DNA recognition helix of mouse Zif268. The PCR products were joined by overlap-PCR and nested PCR, and then inserted into a vector coding the endonuclease domain of FokI nuclease. By these steps, we successfully synthesised intended ZFN vectors containing 4 to 6 fingers. Next, we evaluated the functions of constructed ZFN. The mRNA of constructed ZFN were transcribed in vitro and injected into the cytoplasm of C57BL/6N zygotes. After 24 h of culture, 2-cell stage embryos were subjected to genomic PCR of the target locus, and the PCR products were directly sequenced. When ZFN mRNA for mouse Rosa26 was injected, 3- to 146-bp deletions were detected in 92.8% of injected embryos. This result was almost the same as previously reported for ZFN, indicating that our novel construction method can synthesise functional ZFN, which work as a site-directed nuclease, and that efficiency was comparable with those constructed by conventional PCR methods using long oligonucleotide sets (60 bp).
Production of hypoallergenic milk from DNA-free beta-lactoglobulin (BLG) gene knockout cow using zinc-finger nucleases mRNA