Abstract
The transgenic process allows for obtaining genetically modified animals for divers biomedical applications. A number of transgenic animals for xenotransplantation have been generated with the somatic cell nuclear transfer (SCNT) method. Thereby, efficient nucleic acid delivery to donor cells such as fibroblasts is of particular importance. The objective of this study was to establish stable transgene expressing porcine fetal fibroblast cell lines using magnetic nanoparticle-based gene delivery vectors under a gradient magnetic field. Magnetic transfection complexes prepared by self-assembly of suitable magnetic nanoparticles, plasmid DNA, and an enhancer under an inhomogeneous magnetic field enabled the rapid and efficient delivery of a gene construct (pCD59-GFPBsd) into porcine fetal fibroblasts. The applied vector dose was magnetically sedimented on the cell surface within 30 min as visualized by fluorescence microscopy. The PCR and RT-PCR analysis confirmed not only the presence but also the expression of transgene in all magnetofected transgenic fibroblast cell lines which survived antibiotic selection. The cells were characterized by high survival rates and proliferative activities as well as correct chromosome number. The developed nanomagnetic gene delivery formulation proved to be an effective tool for the production of genetically engineered fibroblasts and may be used in future in SCNT techniques for breeding new transgenic animals for the purpose of xenotransplantation.Electronic supplementary materialThe online version of this article (doi:10.1007/s12033-016-9934-1) contains supplementary material, which is available to authorized users.
Highlights
Since the term ‘‘transgenic’’ was used for the first time [1], there has been a growth in the application of genetically modified animals in diverse biological and medical areas, both for scientific and economic purposes [2,3,4,5]
PEI-Mag2 core–shell-type iron oxide magnetic nanoparticles (MNPs) with a surface coating suitable for nucleic acid delivery formulated from the fluorinated surfactant ZONYL FSA (lithium 3-[2-(perfluoroalkyl)ethylthio]propionate) and 25-kDa branched polyethylenimine (PEI25Br) were used to associate magnetic transfection complexes
This study demonstrates that magnetofection appears to be an alternative method for viral and non-viral gene delivery into porcine fibroblasts
Summary
Since the term ‘‘transgenic’’ was used for the first time [1], there has been a growth in the application of genetically modified animals in diverse biological and medical areas, both for scientific and economic purposes [2,3,4,5]. Somatic cell nuclear transfer (SCNT), known as cloning, holds the greatest promise for significant improvements in the creation of transgenic animals. This method enables cell manipulation in vitro including the addition of a suitable gene or inactivation of an endogenous gene as well as selection of modified cells and their storage for future application. It can result in developing cheaper and easier procedures for transgenic animal production [6]. Despite high transduction efficiency and long-term gene expression, viral vectors suffer from
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