To develop a safe and effective nonviral gene delivery system for transgenic chicken manipulation, we developed gelatin nanocarriers using a reporter plasmid (pEGFP-C1; enhanced green fluorescence protein, EGFP) that expressed EGFP. pEGFP-C1-containing gelatin nanoparticles (GP/pEGFP) were prepared using a water-ethanol solvent displacement method and characterized by size, surface charge, DNA loading, and DNA protection ability. For gene delivery, pEGFP-C1 was stably and efficiently encapsulated in GPs that were approximately 300 nm in diameter with a slight negative surface charge, which was prepared from gelatin solution at pH 8.0. Approximately, 85% of the plasmid DNA was encapsulated in the GPs. Electrophoresis results showed that the GPs provided protection against DNase I digestion. We used the GP/pEGFP as a vector to transfect cells and chicken embryos. The vector was nontoxic to cells, and GFP expression was effectively expressed 24 h after HeLa cell transfection. Direct injection was adapted for vector transport to the chicken embryo; injection in the area opaca (Ao) of the egg resulted in the highest hatching rate without affecting embryo development. GFP gene expression in embryo sections was observed 4 days after injection. The results of this study demonstrate that GPs are a suitable nonviral vector for delivering exogenous genes for transgenic chicken manipulation.
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