Construction Of Magnetic Nanoparticles Gene Delivery System And Application In Gene Delivery

The low efficiency of nonviral gene vectors, the receptor-dependent host tropism of adenoviral orlow titers of retroviral vectors limit their utility in gene delivery. To overcome these deficiencies, weused magnetic nanoparticles(MNPS) as gene vectors to transfer genes, which have some characteristicssuch as can combine with big fragment DNA, low immunogenic, good biocompatibility and targetedgene delivery by application of a magnetic field. Nucleic acids carried by magnetic nanoparticles canoccur directional movement and enrich on the cell surface, which increased contact time and exposuredose of the magnetic nanoparticles/gene complexes with cells. It would contribute to study geneexpression, gene function and targeted gene therapy. In this study, PEI-coated magnetic nanoparticleswere characterized, which were used as gene vector to transfer genes in vivo and in vitro by magneticforce.1.The morphology characteristic, particle size and surface potential of PEI-coated magneticnanoparticles were characterized. The results of electron microscopy and atomic force microscopyshowed that magnetic nanoparticles had100nm diameter with good dispersion, Zeta potential ofmagnetic nanoparticles was29.4mV with positive charge, which can bind with DNA. As the genevector, the DNA binding ability is essential requirement. DNA binding ability of magnetic nanoparticlescan be determined by gel retardation assay, co-sedimentation assay and atomic force microscopy. Theresults proved magnetic nanoparticles had stronger ability to bind DNA. DNA and magneticnanoparticles can form close and tight complex structure. The magnetic nanoparticles can protect DNAagainst the digestion of nuclease. Whether MNPs/DNA complexes influenced cell viability wasinvestigated in several cell lines, including293T, MDCK, Vero and CEF cells by MTT assays.MNPs/DNA complexes showed no obvious negative effect on cell viability. The result of magneticnanoparticles transfection into293T cells showed that the magnetic nanoparticles had high transfectionefficiency, and the highest transfection efficiency was91%when the ratio of magnetic nanoparticles andDNA was1:1, higher than the positive control of lipofectamine transfection efficiency (86%).2.In the present study, the2A region of foot-and-mouth disease virus (FMDV) was utilized toconstruct a bicistron vector, in which tandem retrocyclin2(RC2) gene and dTomato gene were fusedinto a single open reading frame linked by FMDV2A region. The fused genes were placed under thecontrol of CMV promoter in pCDNA4/TO EGFP vector to construct the recombinant expressionplasmids pCDNA4-dTomato-2A-3R2/6R2. The plasmids pCDNA4-dTomato-2A-3R2/6R2wastransfected into293T cells mediated by MNPs with application of a magnetic field. The results showedthat the fused genes of red fluorescent protein dTomato and muti-copy RC2linked by2A region offoot-and-mouth disease virus were expressed in293T successfully. The2A region mediated aco-translational cleavage event at its own carboxyl-terminus resulting in the release of each individualprotein product. Efficient cleavage was observed and all two proteins were functional, coexpression ofdTomato enabled us to track gene expression in293T cells conveniently. The tandem retrocyclin2can potently inhibit H5N1highly pathogenic avian influenza virus production in293T cells, which providea basis for further development of retrocyclin2for prophylaxis and therapy of H5N1virus infection inhumans.3.The chicken embryo fibroblast(CEF) cells isolated from the chicken embryo were cultured invitro to construct a cell line. As the exogenous gene the plasmid pEGFP-N1was used to transfect CEFcells mediated by MNPs. G418was employed to screen the drug-resistance cell clones to obtain stablecell lines expressing green fluorescent protein, which laid the foundation for the study of transgenicchickens.4.The plasmids pCDNA4-dTomato-2A-3R2/6R2were transfected into CEF cells and chickenembryo mediated by MNPs with application of a magnetic field. The results showed that the fusedgenes of red fluorescent protein dTomato and tandem RC2linked by2A region of foot-and-mouthdisease virus were expressed in CEF cells and chicken embryo successfully. The tandem retrocyclin2can potently inhibit H5N1highly pathogenic avian influenza virus production in CEF cells and chickenembryo, which provide a basis for further development of application of MNPs to gene delivery in vitroand in vivo, retrocyclin2for prophylaxis and therapy of H5N1virus infection in poultry, producingtransgenic chickens with suppression of avian influenza transmission.