Study On Genetic Transformation Of Animals With Nano Gene Vector

Over the past30years, the research of transgenic technology in animal genetic breeding and newbreed selecting had achieved noticeable advancement in biology and zoology. There are several maintraditional methods of producing transgenic animals,such as retrovirus mediated method, embryonicstem cells and Microinjection.But these methods had limited application and defects in the research ofanimal genetic breeding in the future. Sperm-mediated gene transfer approach is a promising transgenicmethod.It has many merits,such as cheap costs, hight efficiency,simplicity,not require costly equipmentet al. Meanwhile,nanoparticles have been applied in gene transformation,which is one of the majorachievements that have been gained in nano-biotechnology field. The magnetic nanoparticles have somecharacteristics such as have a high carrying efficiency,without immunogenic, and high targeting undermagnetic field ect.All these advantages provide new approach and ideas for the animal breeding.Thispaper has described the systematic researches of PEI modified magnetic nanoparticles as animaltransgenic carrier, and a series of experiments using nanoparticles were carried out for transgenicanimals.Three parts were included in the studies:1. Preparation and Characterization of PEI-modified magnetic nanoparticles gene carrierThe morphology characteristic of PEI-modified magnetic nanoparticles were characterized byscanning electron microscopy，transmission electron microscope and atomic force microscope.It hasspherical shape with the average diameter about100nm，good dispersion. It was found that the surfaceof the particles becomes rough and coarse with increased average diameter, which implied the effectiveconjugating between magnetic nanoparticles with plasmid DNA,and the average diameter increases to130nm after conjugated with the plasmid DNA. The Agarose gel electrophoresis experiments show thatplasmids DNA and PEI magnetic nanoparticles can form close and tight complex structure,and plasmidsDNA can be protected effectively against degradation of endonuclease and exonuclease by PEI-modifiedmagnetic nanoparticles, the results showed. Meanwhile, We confirm that the most optimal mass ratio ofnanoparticle/DNA is3:1by conducting a series of experiments.2. Genetic transformation of mice with nanoparticle gene vectorTo evaluate the performance of the magnetic nanoparticles as gene transfer vector for breedingtransgenic animals, we investigated a new approach to deliver green fluorescent protein (GFP) gene toPK-15cell using PEI-modified magnetic nanoparticles as gene vector. Respectively,PEI-modifiedmagnetic nanoparticles and liposomes as a control group were carried out for transformation experimenton PK-15.The results illuminated that PEI-modified magnetic nanoparticles could transfer the GFP geneinto the PK-15cells and expressed green fluorescence protein, and the transformation efficiency wasalmost28.23％, which signifcantly higher than that of the liposomes group(15.42%) and controlgroup(3.79%). Moreover,the positive rate of co-transfection of GFP and DsRed plasmids usingPEI-modified magnetic nanoparticles was6.85%. In conclusion, this work provides a new approach todeliver genes to PK-15cells using PEI-modified magnetic nanoparticles as gene transfer vector. The achieving of gene delivery into PK-15by using magnetic nanoparticles provides an importantexperimental basis for the application of the magnetic nanoparticles as gene transfer vector forsperm-mediated gene transfer.3. Genetic transformation of mice with nanoparticle-sperm-mediated gene transferThe sperm-mediated gene transfer method is applied in many species that use spermatozoa forreproduction recently, which has been shown various results. In our study, we show that transgenic micecan be efficiently produced by employing a simple transfection method that uses magnetic nanoparticles.Using confocal microscopy, fluorophore-labelled nano gene vector were detected on external surfaces ofthe spermatozoa membrane.The cytotoxicity of magnetic nanoparticles to spermatozoa was tested bydouble staining with SYBR-14/PI and flow cytometry. The t PEI-modified magnetic nanoparticles andliposomes had low cytotoxicity, with53.64%,61.62%cell viability respectively.Genomic DNA wasextracted from F1transgenic mice tails.PCR were applied to identify transgenic mice.The positive rateof PCR for GFP in transgenic mice was62.5%after the SMGT was optimized by nano genevector,which was significantly higher than that of the liposomes group(50%)and control group(0%)．Taken together, our results clearly demonstrate that the complexes formed between magneticnanoparticles and plasmid DNA were combined to spermatozoa at a higher efficiency,compared tomethods that using lipofection or DNA alone,and the exogenous DNA can be delivered into the oocytethrough IVF,therefore, magnetic nanoparticles gene vectors could help improve the efficiency of SMGT.