Construction And Activity Validation Of ZFN And TALEN For Targeting Pig’s ApoE Gene

The homologous recombination and viral-mediated gene transfer are two commonlyused methods in animal genome engineering. The extreme low efficiency and poor specificityhampered their application in generation of transgenic animals and human gene therapy. Zincfinger nucleases（ZFNs）, a newly developed technology, targeted genome engineering, showspromise in improving the efficiency of gene targeting by introducing DNA double strandbreaks（DSBs）in target genes. Custom-designed zinc finger nucleases（ZFNs）combine thenon-specific cleavage domain（N）of Fok I restriction endonuclease with zinc finger proteins（ZFPs）. TALE nucleases（TALENs）are another new technology to specifically andeffectively modify genome following zinc figure nuclease technology established. TALENtechnology is based on the discovery of TALE（stranscription activator–like effectors）secretedby plant pathogen Xanthomonas. The DNA binding domain of TALEs fused to the Fok Icleavage domain can be used to generate chimeric nucleases（TALENs）that bind to the DNAand create double strand breaks（DSBs）. It has been demonstrated that artificial TALENs canspecifically and effectively modify genome. The activities of TALENs have been tested inyeast, plant and mammalian cells including human cells.Apolipoprotein E（ApoE）is a polymorphic protein. ApoE gene can regulate manybiological functions. It has been reported that the defection or mutation of ApoE gene relateswith coronary heart disease, Alzheimer’s disease, and aging disease, leading to the serioushypercholesterolemia and atherosclerotic arterial disease. Because of the high homologousbetween human and swine, the ApoE gene of swine, taken as the target gene, is modified byZFN and TALEN technology. The modification efficiencies between the two methods arecompared. The results will provide great help for constructing efficient transgenic method anddisease model production. The results are as follows:（1） Two pairs of ZFNs targeting ApoE gene of pig were designed by CoDA method.The eukaryotic expression vector was constructed and ZFNs’ activity was identified by yeasttwo-hybrid analysis. The constructed vectors were transformed to HEK293cells to detect theactivity. ZFNs worked well.（2） At the same region of ZFN targeting sites, TALEN recognizing12nucleotides of ApoE gene was designed and obtained by PCR and digestion-ligation methods. Theconstructed TALEN and pST1374vector were linked to construct the eukaryotic expressionvector. The TALEN expression vectors and reporter vector were co-transformed to HEK293cells to detect the activity. TALEN operated successfully.