Design, construction, and analysis of specific zinc finger nucleases for microphthalmia - associate transcription factor

This work studied the design, construction, and cleavage analysis of zinc finger nucleases (ZFNs) that could cut the specific sequences within microphthalmia - associate transcription factor (mitfa) of zebra fish. The target site and ZFPs were selected and designed with zinc finger tools, while the...

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Bibliographic Details
Published in:Brazilian archives of biology and technology 2012-08, Vol.55 (4), p.559-568
Main Authors: Wang, Wenwen, Liu, Hongmao, Zhao, Haibin, Luo, Zaili, Shi, Yongxiang
Format: Article
Language:English
Subjects:
DNAWorks
gene knockout
gene synthesis
Zinc finger nuclease
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Summary:This work studied the design, construction, and cleavage analysis of zinc finger nucleases (ZFNs) that could cut the specific sequences within microphthalmia - associate transcription factor (mitfa) of zebra fish. The target site and ZFPs were selected and designed with zinc finger tools, while the ZFPs were synthesized using DNAWorks and two-step PCR. The ZFNs were constructed, expressed, purified, and analyzed in vitro. As expected, the designed ZFNs could create a double-stand break (DSB) at the target site in vitro. The DNAWorks, two-step PCR, and an optimized process of protein expression were firstly induced in the construction of ZFNs successfully, which was an effective and simplified protocol. These results could be useful for further application of ZFNs - mediated gene targeting.
ISSN:1516-8913
1516-8913
1678-4324
DOI:10.1590/S1516-89132012000400011