Construction Of A System For ZFN Technology, And For Improving The Efficiency Of The Gene Targeting On Transgenic Animals

The foreign gene can be integrated into the targeted site of genome, and it has a highefficiency using gene targeting technology with ZFN (zinc finger nuclease) technologytogether. It will promote the research of transgenic animal. Our experiment constructed ascreen system for artificial zinc-finger proteins and a new method for construction of randomArtificial Zinc-finger Proteins library. We screened and constructed zinc finger nucleases forβ-casein using our system. Then，the zinc finger nuclease was used for gene targetingresearch，the results show that it can improve the efficiency of gene targeting integration,more than5-10times.1. Construction of a screen system for Artificial Zinc-finger Proteins: The system has areporter vector (pReport-LacZ), a random Artificial Zinc-finger Proteins expression vector(pBD-Gal11p-ZFS) and a activation vector (PAD-of Gal4). The reporter vectorhas ampicillin (Amp) resistance, two replication status that can be regulated, multiplecopies (20-50/cell) and single-copy the (1-2/cell). β-galactose enzyme gene (LacZ) forreporter and aminoglycoside adenosine acyltransferase gene (aada,sm) for screen areexpressed by the same promoter element; The random Artificial Zinc-finger Proteinsexpression vector has chloramphenicol resistance (Cm), and expressed prokaryoticrandom zinc finger fragment; The activetion vector has a kanamycin resistance (Kan), andexpressed activation domain (RNAPα-Gal4).2. Construction of random artificial zinc-finger library: Random artificial Zinc-fingerfragment was got by random primers and overlapping PCR. Then, random ArtificialZinc-finger library was built by PCR amplification, restriction enzyme digestion, ligation,electrotransformation and plasmid prep. The library has more than1.48×1021(1518)different Artificial Zinc-finger.3. Screen for Artificial Zinc-finger: Best conditions of the screen system for ArtificialZinc-finger Proteins was determined by multiple comparison tests. We screened the zincfinger protein for β-casein using the screen system for Artificial Zinc-finger Proteins. Theactivation of ZFP was tested by high-throughput β-galactosidase assay.4. The detection for efficiency of gene targeting integration: In this study, quantitativePCR and monoclonal screening method was used for detection, the results show ZFN can improve the efficiency of gene targeting integration.In summary, we successfully constructed a screen system for Artificial Zinc-finger proteinsand random Artificial Zinc-finger Proteins library. The method for construction of randomArtificial Zinc-finger Proteins library has some advantages, such as: need less time, cost lowand convenient. Any biological molecular lab can complete, will be widely applied. Bestconditions of the Artificial Zinc-finger Proteins screening was determined by multiplecomparison tests. Four pairs of zinc finger nuclease eukaryotic expression vector weresuccessfully constructed for a locus for β-casein gene and studied the impact on the efficiencyof gene targeting integration. The results show that the selected zinc finger nucleases couldincrease the integration efficiency of gene targeting.