title: A novel genetic system based on zinc finger nucleases for the identification of interactions between proteins in vivo.
abstract: Yeast two-hybrid (Y2H) methods are powerful tools for detecting protein-protein interactions. The traditional Y2H method has been widely applied to screen novel protein interactions since it was established two decades ago. The high false-positive rate of the traditional method drove the development of modified Y2H systems. Here, we describe a novel Y2H system using zinc-finger nucleases (ZFNs). ZFNs contain two functional domains, a zinc-finger DNA-binding domain (ZFP) and a non-specific nuclease domain (FokI). In this system, the bait is expressed as a fusion protein with a specific ZFP, and the prey is fused to the FokI. A reporter vector is designed such that the ZFN target site disrupts the Gal4 open reading frame. By transforming the three plasmids into a yeast strain (AH109), the interaction between the bait and prey proteins reconstitutes ZFN function and generates the double-strand break (DSB) on its target site. The DNA DSB repair restores Gal4 function, which activates the expression of the four reporter genes. We used p53-SV40LT interacting proteins to prove the concept. In addition, 80% positive rate was observed in a cDNA screening test against WDSV orfA protein. Our results strongly suggested that this Y2H system could increase screening reliability and reproducibility, and provide a novel approach for interactomics research.
authors.affiliation: College of Animal Science & Technology, Shaan'xi Key Laboratory of Molecular Biology for Agriculture, Northwest A&F University, YangLing, Shaan'xi, P. R. China.
authors.forename: Ling
authors.initials: L
authors.lastname: Wang
authors.affiliation: College of Animal Science & Technology, Shaan'xi Key Laboratory of Molecular Biology for Agriculture, Northwest A&F University, YangLing, Shaan'xi, P. R. China.
authors.forename: Kun
authors.initials: K
authors.lastname: Xu
authors.affiliation: College of Animal Science & Technology, Shaan'xi Key Laboratory of Molecular Biology for Agriculture, Northwest A&F University, YangLing, Shaan'xi, P. R. China.
authors.forename: Juan
authors.initials: J
authors.lastname: Lin
authors.affiliation: College of Animal Science & Technology, Shaan'xi Key Laboratory of Molecular Biology for Agriculture, Northwest A&F University, YangLing, Shaan'xi, P. R. China.
authors.forename: Simin
authors.initials: S
authors.lastname: Shao
authors.affiliation: College of Animal Science & Technology, Shaan'xi Key Laboratory of Molecular Biology for Agriculture, Northwest A&F University, YangLing, Shaan'xi, P. R. China.
authors.forename: Tingting
authors.initials: T
authors.lastname: Zhang
authors.affiliation: College of Animal Science & Technology, Shaan'xi Key Laboratory of Molecular Biology for Agriculture, Northwest A&F University, YangLing, Shaan'xi, P. R. China.
authors.forename: Huarong
authors.initials: H
authors.lastname: Xu
authors.affiliation: College of Animal Science & Technology, Shaan'xi Key Laboratory of Molecular Biology for Agriculture, Northwest A&F University, YangLing, Shaan'xi, P. R. China.
authors.forename: Zehui
authors.initials: Z
authors.lastname: Wei
authors.affiliation: College of Animal Science & Technology, Shaan'xi Key Laboratory of Molecular Biology for Agriculture, Northwest A&F University, YangLing, Shaan'xi, P. R. China.
authors.forename: Zhiying
authors.initials: Z
authors.lastname: Zhang
mesh_terms: D000818:Animals; D003851:Deoxyribonucleases / Q000737:chemistry* / Q000378:metabolism*; D015723:Gene Library; D017930:Genes, Reporter / Q000235:genetics; D051379:Mice; D011485:Protein Binding; D020798:Two-Hybrid System Techniques; D016335:Zinc Fingers
chemical_list: D003851:Deoxyribonucleases