Method Study On Magnetic Nanoparticles As Gene Engineering Carriers In Cell Transformation

This research has used magnetic nanoparticles as gene engineering carrier to conduct transgene research in animal and plant cell. We have chosen the polyethylenamine (PEI) magnetic nanoparticles, Amine silane magnetic nanoparticles and chitosan magnetic nanoparticles as gene carrier. In this paper, we have carried out the research on their morphology characteristic, the transfection efficiency in animal and plant cell and explored its possible influential factors. The main research results are as follows:(1) The morphology characteristic of the three kinds of magnetic nanoparticles were characterized by scanning electron microscopy, these magnetic nanoparticles were spherical. The diameter of PEI magnetic nanoparticles is 100-200nm, with good dispersion. Amine silane magnetic nanoparticles and chitosan magnetic nanoparticles were about 50nm in diameter, with serious aggregation phenomenon. The gel retardation assay had proved these magnetic nanoparticles can bind to plasmid DNA. The PEI magnetic nanoparticles had high DNA-binding capacity, but the amine silane magnetic nanoparticles and the chitosan magnetic nanoparticles had relatively lower DNA-binding capacity. Further researches had been conducted to analyze the combination of PEI magnetic nanoparticles and plasmid DNA by atomic force microscope and cirular dichroism, the results showed DNA and PEI magnetic nanoparticles can form close and tight complex structure. The complex of magnetic nanoparticles and DNA were digested by DNase I, the results showed the PEI magnetic nanoparticles can protect DNA from digesting by DNase I, but amino magnetic nanoparticles and chitosan magnetic nanoparticles didn't show the ability of protecting DNA from digesting by DNase I.(2) The cytotoxicity of magnetic nanoparticles to PK-15 cell was tested by MTT assay. The three kinds of magnetic nanoparticles had low cytotoxicity, with 68%,83%,87% cell viability respectively. The PEI magnetic nanoparticles had the highest cytotoxicity, and the amine silane magnetic nanoparticles and the chitosan magnetic nanoparticles had relatively lower cytotoxicity. The results of magnetic nanoparticles transfection in PK-15 cells showed that the PEI magnetic nanoparticles had high transfection efficiency, and the highest transfection efficiency was 25% when the quality ratio of magnetic nanoparticles and DNA was 1:1, much higher than the positive control of lipofectamine transfection efficiency (10%). And the chitosan magnetic nanoparticles transfection efficiency was much lower, about 3%. There was no gene expression result in PK-15 cells transferred by the amine silane magnetic nanoparticles. The results showed the PEI magnetic nanoparticle was a good gene engineering carrier in PK-15 cells.(3) We first used magnetic nanoparticles as gene engineering carrier to carry DNA into arabidopsis protoplast successfully. It was found that the PEI magnetic nanoparticles had high cytotoxicity, and the cell viability was only 10% when the amount of the PEI magnetic nanoparticles was 10μg. The amine silane magnetic nanoparticles and the chitosan magnetic nanoparticles had relatively lower cytotoxicity, with cell viability above 74% and 81% respectively. The results of magnetic nanoparticles transformation in arabidopsis protoplast cells showed that there was positive effect with PEI magnetic nanoparticles transformation, but the transformation efficiency was very low. When the quality ratio of PEI magnetic nanoparticles and DNA was 1:2, the transformation efficiency was the highest, about 11%, much lower than the positive control transformation efficiency of PEG (about 65%). But there were no GFP expression in arabidopsis protoplast with the amine silane magnetic nanoparticles and the chitosan magnetic nanoparticles transformation.