Construction Of A Screen System For Artificial Zinc-finger Proteins And Application For Bovine3Casein Gene

Zinc-finger nuclease is one kind of artificial engineering restriction enzyme. Thesenucleases created by fusing an engineered ZF DNA binding domain to the DNA cleavagedomain of the FokI type IIS restriction enzyme and were widely used in genes modificationand genes therapy of animals and plants. However, the off-target effects of ZFNs inaccuracyincision, caused by activity of ZFPs, generate cytotoxicity and genotoxicity. So ZFPs whichare high specificity and affinity, play a key role in application of ZFNs. This study basing ontheory of bacteria two-hybrid screening system, construct a screen system for artificialzinc-finger proteins and application for bovine casein gene. Using the screen system, wescreened the artificial zinc-finger proteins that target bovine casein gene, and confirmed theactivity of candidate proteins using the Yeast One Hybrid system. This work shows a novelapproach for screening of zinc-finger proteins, and lay basis for the application of zinc-fingernucleases in targeting bovine casein gene.The results of this experiment as follows.1.The experiment was successfully constructed a new zinc finger protein screeningsystem,which include reporter plasmid pReport-N-EGFP, activator plasmide pAD-R-G andthe random zinc-finger protein library pBD-P-Gal11P-ZFS.Designed random zinc fingersequence,basing on characteristic of efficiency zinc finger protein and degeneracy of protein,was cloned into pBD-P-Gal11P through BbsI restriction enzyme sites and finally got randomzinc finger protein pool. pReport-N-EGFP contains basic promoter, single copy controllingelement, medium copy controlling element, reporter gene EGFP and resistance gene aadadownstream of EGFP, two restriction enzyme Bsu36I which provide for the insertion of targetsite. pReport-N-EGFP is maintained in the single copy state by using the oriS and repEelements together with the parABC partition determinants. The medium copy state is obtainedby employing trfA replicator acting at oriV. Activated plasmid contain RNA polymerase α andthe transcription activation domain of Gal4.2.Screening results aim to target site of bovine beta-casein.We software designed9nucleotide specificity zinc finger binding site in casein gene of bovine through ZiFi andconstructed pReport-N-EGFP-CNS by insertion the half zinc finger binding site into vector pReport-N-EGFP through two Bsu36I site. Then we cotransform host strain DH5α withpReport-N-EGFP-CNS,pAD-RNAP-alpha-GAL4and random zinc-finger protein poolpBD-P-Gal11P-ZFS to screen coactions of zinc finger protein and zinc finger binding site.Binding of a Gal11P–zinc-finger array hybrid protein to a target binding site leads torecruitment of RNA polymerase complexes, which have incorporated an RNA polymerasea-subunit–Gal4hybrid protein to a proximal promoter. This recruitment occurs as a result ofinteraction between the Gal11P and Gal4proteins and results in increased transcription ofreporter gene(s) downstream of the promoter. Failure of a Gal11P–zinc-finger array hybridprotein to bind a target site results in noactivated expression of the reporter gene. Theconfirmed screening conditions of this experiment are kanamycin30ug/ml, chloramphenicol34ug/ml, Ampicillin50ug/ml, streptomycin80ug/ml0.2%gluclose and IPTG50uM. GFPfluorescence intensity of the experimental group enhanced100-250times relative to control.3. We confirmed the activity of candidate proteins using the Yeast One Hybrid system.Frist, we construct bait-reporter vector pLaczi-1z,pLaczi-2z, pLaczi-3z,pLaczi-1y, pLaczi-2y,pLaczi-3y including9nucleotide specificity zinc finger binding site of bovine casein gene,and we generate bait-reporter strains by homologous integrate linearized bait-reporterplasmids into Y1HGold,respectively. Second,we construct the prey plasmid pGADT7-ZFincluding candidate proteins we have got through screening system. Third, using the protocolof the Yeast Transformation System2, the activation vector pGADT7-ZF transferred to theyeast reporter strain relative to the binding site and the plasmid encoding interacting proteincan grow on Leu-deficient medium. Then, we tested LacZ activity of colonies growed onLeu-deficient medium using method of ONPG, further defined the activity of zinc fingerproteins obtained by the screening systerm.