Journal article
Targeted editing of goat genome with modular-assembly zinc finger nucleases based on activity prediction by computational molecular modeling
College of Animal Science and Technology, Nanjing Agricultural University, Nanjing, 210095, People's Republic of China.1
Zinc finger nuclease (ZFN) technology can mediate targeted genome modification to produce transgenic animals in a high-efficient and biological-safe way. Modular assembly is a rapid, convenient and open-source method for the synthesis of ZFNs. However, this biotechnology is hampered by multistep construction, low-efficiency editing and off-target cleavage.
Here we synthesized and tested six pairs of three- or four-finger ZFNs to target one site in goat beta-lactoglobulin (BLG, a dominant allergen in goat milk) gene. Homology modeling was applied to build the structure model of ZFNs to predict their editing activities targeting at goat BLG gene. Goat fibroblast cells were transfected with plasmids that encoded ZFN pairs, and genomic DNA was isolated 72 h later for genome editing efficiency assay.
The results of editing efficiency assay demonstrated that ZFNs with optimal interaction modes can edit goat BLG gene more efficiently, whereas ZFNs with unexpected interaction modes showed lower activities in editing BLG gene. We concluded that modular-assembly ZFNs can provide a rapid, public-available, and easy-to-practice platform for transgenic animal research and molecular modeling would help as a useful tool for ZFNs activity prediction.
Language: | English |
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Publisher: | Springer Netherlands |
Year: | 2013 |
Pages: | 4251-4256 |
Journal subtitle: | An International Journal on Molecular and Cellular Biology |
ISSN: | 15734978 and 03014851 |
Types: | Journal article |
DOI: | 10.1007/s11033-013-2507-5 |
Amino Acid Sequence Animal Anatomy / Morphology / Histology Animal Biochemistry Animals BLG Base Sequence Biomedical and Life Sciences Computational Biology Endonucleases Enzyme Activation Genome Genome editing Genomics Goat Goats Life Sciences Models, Molecular Modular-assembly Molecular Sequence Data Molecular modeling Protein Conformation Sequence Alignment ZFN Zinc Fingers