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Generation of Transgenic Porcine Fibroblast Cell Lines Using Nanomagnetic Gene Delivery Vectors.

Grześkowiak BF, Hryhorowicz M, Tuśnio K, Grzeszkowiak M, Załęski K, Lipiński D, Zeyland J, Mykhaylyk O, Słomski R, Jurga S, Woźniak A - Mol. Biotechnol. (2016)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: The NanoBioMedical Centre, Adam Mickiewicz University, Umultowska 85, 61-614, Poznan, Poland. bartoszg@amu.edu.pl.

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.

No MeSH data available.


Related in: MedlinePlus

Characteristics of PEI-Mag2 magnetic nanoparticles used to assemble magnetic lipoplexes. a, b High-resolution transmission electron microscopy images. Spherical core shape and crystalline structure of the particles are clearly visible. c The size distribution of the particles based on quantitative analysis of the TEM images. d Scanning electron microscopy images demonstrating the spherical shape of the particles. e Magnetization curve displaying magnetic properties
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Fig2: Characteristics of PEI-Mag2 magnetic nanoparticles used to assemble magnetic lipoplexes. a, b High-resolution transmission electron microscopy images. Spherical core shape and crystalline structure of the particles are clearly visible. c The size distribution of the particles based on quantitative analysis of the TEM images. d Scanning electron microscopy images demonstrating the spherical shape of the particles. e Magnetization curve displaying magnetic properties

Mentions: 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 (PEI-25Br) were used to associate magnetic transfection complexes. The size, morphology, ζ-potential, as well as magnetic properties of these particles were assessed. The particle sizes and morphology were evaluated by electron microscopy. The TEM micrographs show the presence of apparently spherical iron oxide cores (Fig. 2a, b) and quite uniform size distributions with the core size being of 8.0 ± 2.2 nm (Fig. 2c). The SEM image demonstrates the spherical shape as well as multicore structure of the MNPs (Fig. 2d). When drying, particles tend to agglomerate due to their high surface energy. Figure 2e presents the magnetic properties of PEI-Mag2 particles. The saturation magnetization registered at room temperature was 56 emu/g iron. The magnetization curve exhibited a superparamagnetic behavior due to zero coercivity and zero remanence. The mean hydrodynamic diameter measured in aqueous suspension was 28 nm, and the electrokinetic potential was highly positive at +48.8 ± 1.0 mV resulting from the presence of PEI in the surface layer of the particles.Fig. 2


Generation of Transgenic Porcine Fibroblast Cell Lines Using Nanomagnetic Gene Delivery Vectors.

Grześkowiak BF, Hryhorowicz M, Tuśnio K, Grzeszkowiak M, Załęski K, Lipiński D, Zeyland J, Mykhaylyk O, Słomski R, Jurga S, Woźniak A - Mol. Biotechnol. (2016)

Characteristics of PEI-Mag2 magnetic nanoparticles used to assemble magnetic lipoplexes. a, b High-resolution transmission electron microscopy images. Spherical core shape and crystalline structure of the particles are clearly visible. c The size distribution of the particles based on quantitative analysis of the TEM images. d Scanning electron microscopy images demonstrating the spherical shape of the particles. e Magnetization curve displaying magnetic properties
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License
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getmorefigures.php?uid=PMC4829627&req=5

Fig2: Characteristics of PEI-Mag2 magnetic nanoparticles used to assemble magnetic lipoplexes. a, b High-resolution transmission electron microscopy images. Spherical core shape and crystalline structure of the particles are clearly visible. c The size distribution of the particles based on quantitative analysis of the TEM images. d Scanning electron microscopy images demonstrating the spherical shape of the particles. e Magnetization curve displaying magnetic properties
Mentions: 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 (PEI-25Br) were used to associate magnetic transfection complexes. The size, morphology, ζ-potential, as well as magnetic properties of these particles were assessed. The particle sizes and morphology were evaluated by electron microscopy. The TEM micrographs show the presence of apparently spherical iron oxide cores (Fig. 2a, b) and quite uniform size distributions with the core size being of 8.0 ± 2.2 nm (Fig. 2c). The SEM image demonstrates the spherical shape as well as multicore structure of the MNPs (Fig. 2d). When drying, particles tend to agglomerate due to their high surface energy. Figure 2e presents the magnetic properties of PEI-Mag2 particles. The saturation magnetization registered at room temperature was 56 emu/g iron. The magnetization curve exhibited a superparamagnetic behavior due to zero coercivity and zero remanence. The mean hydrodynamic diameter measured in aqueous suspension was 28 nm, and the electrokinetic potential was highly positive at +48.8 ± 1.0 mV resulting from the presence of PEI in the surface layer of the particles.Fig. 2

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: The NanoBioMedical Centre, Adam Mickiewicz University, Umultowska 85, 61-614, Poznan, Poland. bartoszg@amu.edu.pl.

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.

No MeSH data available.


Related in: MedlinePlus