The Nuclear Donor Cell Lines Of Bovine FMDV Receptor Integrin β6Knocked Out Are Obtained By ZFN Technology

Foot-and-mouth disease (FMD) is a FMD virus (FMDV) which caused an acute,febrile, highly contagious disease in the cattle, pigs, sheep and other cloven hoofedanimal. The Office International des Epizooties (OIE) ranks the FMD in the first paceof Class I deadly contigious diseases. Once it breaks out, all of infected animal andthe animal contacted with infected animal must be killed. This will not only causehuge economic losses, but also serious harm to the development of animal husbandry.At present, the effective precautionary measure for preventing FMD is toimplement vaccine injection in most popular FMD countries. However, FMDV has7serotypes and fast to variate, no cross immunity reaction between different serotypevaccines.The vaccine method is difficult to control and eradicate FMD,so thescientists began to concern the relationship between virus and host when they focusedon vaccine development at the same time, especially the effect of virus receptor in theprocess of infection.They wanted to block viral infection by knocking out or silencingthe key receptor, so as to achieve the purpose of controlling and eradicating FMD.Research has showed that the integrin αvβ6is the key receptor of FMDV causinginfection and disease which could combine with all serotypes of FMDV. Transgenicmice knocked out αv gene led to high mortality rate of the mouse embryos, abnormalgrowth and development and prone to cancer, therefore, the αv gene is not idealreceptor gene for FMD research; β6is a decisive receptor subunit to start infectedwith virulent FMDV, transgenic mouses knocked out integrin β6subunit gene werenormal growth and development. Therefore, knockout of integrin β6is the keyapproach to inhibit virus infection, providing the drug targets for the control anderadication of FMD.The traditional rate of is time-consuming and inefficient.Therefore, in recent years, people constantly tried to find new gene targetingtechnologies for genetic research, the zinc finger nuclease has become an efficient,powerful tool for the genetic operation in the cells and the individual level and successfully applied to a variety of animal.A45-days fetus of Holstein cow were taken out using the operation method in thisstudy, primary bovine fetal fibroblast cell lines were cultured by collagenase IVdigestion, and the F0and F1generation were frozen for subsequent experiments.Primers were designed according to the integrin β6sequence of bovine publishedon NCBI. β6gene was amplified and sequenced using the DNA of primary bovinefetal fibroblasts as template,the results of sequencing were submitted to SIGMAcompany in order to customize the zinc finger nuclease.The electric transfectionprocedures were optimizated and U-023was chosen for primary bovine fetalfibroblasts. ZFNs were transfected into primary bovine fetal fibroblasts using theprogram, T-A clone, DNA sequencing and comparing with the wild type sequence, thenumber of bacterial clone mutation were determined,mutations/clones sequenced isthe targeting efficiency,about22.9%. Monoclonal cells were obtained using limiteddilution method and cloning rings.25positive cell clones were determined by themethod of targeting efficiency of detection.The mutations were categorized into threegroups including deletions (17/25;68%)comprised with8homozygous single-cell clonesand9heterozygous single-cell clones, insertions (2/25;8%)comprised with1homozygoussingle-cell clones and1heterozygous single-cell clones and complex types containing bothdeletions and insertions (6/25;24%) comprised with6heterozygous single-cell clones.The donor vector pβ6LGNR of homologous targeting, targeting for the β6locus，containing GFP-less promoter gene. Homologous targeting vector was linearized andcotransfected into primary bovine intestinal epithelial cells with ZFNs, the expressionof GFP were observed under inverted fluorescence microscope and confirmed ZFNsmediated homologous recombination occured, there were no GFP observed when thepβ6LGNR plasmids were transfected only. Then pβ6LGNR and ZFNs werecotransfected into primary bovine fibroblasts, screened10days by G418,10positivecell clones were obtained using PCR, containing1homozygous and9heterozygous,ZFNs mediated homologous targeting efficiency is3.6%, there were no positive cellclones were obtained when the pβ6LGNR plasmids were transfected only. This studyprovides a drug target for anti FMD, and lays the foundation for the mechanism research of FMD infection and the insertion of FMD other functional genes.