A magnetic nanoparticle-based multiple-gene delivery system for transfection of porcine kidney cells.

Yan Wang,Haixin Cui,Kui Li,Changjiao Sun,Jinhui Cui,Xiang Zhao,Wenjie Chen,Wei Du,Stefan Wölfl

doi:10.1371/journal.pone.0102886

Abstract

Superparamagnetic nanoparticles are promising candidates for gene delivery into mammalian somatic cells and may be useful for reproductive cloning using the somatic cell nuclear transfer technique. However, limited investigations of their potential applications in animal genetics and breeding, particularly multiple-gene delivery by magnetofection, have been performed. Here, we developed a stable, targetable and convenient system for delivering multiple genes into the nuclei of porcine somatic cells using magnetic Fe3O4 nanoparticles as gene carriers. After surface modification by polyethylenimine, the spherical magnetic Fe3O4 nanoparticles showed strong binding affinity for DNA plasmids expressing the genes encoding a green (DNAGFP) or red (DNADsRed) fluorescent protein. At weight ratios of DNAGFP or DNADsRed to magnetic nanoparticles lower than or equal to 10∶1 or 5∶1, respectively, the DNA molecules were completely bound by the magnetic nanoparticles. Atomic force microscopy analyses confirmed binding of the spherical magnetic nanoparticles to stretched DNA strands up to several hundred nanometers in length. As a result, stable and efficient co-expression of GFP and DsRed in porcine kidney PK-15 cells was achieved by magnetofection. The results presented here demonstrate the potential application of magnetic nanoparticles as an attractive delivery system for animal genetics and breeding studies.

Highlights

Transgenic technology has attracted the attention of scientists in various fields, including medicine [1,2,3,4], agriculture [5,6,7,8] and biology [9,10,11,12]
Two plasmids containing the gene encoding a green (GFP; DNAGFP) or red (DsRed; DNADsRed) fluorescent protein were used as markers
Migration of DNAGFP in the gel was retarded at DNA:MagNP ratios of 10:1 and lower (Figures 3a and 3b), and migration of DNADsRed was retarded at ratios of 5:1 and lower (Figures 3c and 3d), indicating the formation of MagNP-DNA complexes at these ratios

Summary

Introduction

Transgenic technology has attracted the attention of scientists in various fields, including medicine [1,2,3,4], agriculture [5,6,7,8] and biology [9,10,11,12]. The process of introducing foreign DNA into host cells, is a necessary step in the genetic modification of crops and livestock. The bottleneck in the success of genetic transformation has been the development of a safe, stable and efficient gene delivery system [13]. Over the past few decades, many different gene delivery methods have been developed for various types of animal and plant cells. Virus-mediated gene delivery utilizes the ability of a virus to inject its DNA into a host cell. Nonviral carriers, which have advantages such as minimal host immune response, stability in storage, and relative ease of production and scale-up, are being developed as alternative methods of gene delivery [19,20]. Non-viral approaches include agrobacterium-mediated methods, chemical methods such as lipofection, and physical methods such as microinjection, gene guns, impalefection, hydrostatic pressure, electroporation, continuous infusion, and sonication

Methods

Results

Discussion

Conclusion