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

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.

Keywords: Magnetic nanoparticles; Magnetofection; Nucleic acid delivery; Porcine fetal fibroblasts; Transgenesis.

Fig. 1

Schematic presentation of the magnetic nanoparticle-mediated generation of transgenic pigs. After formulation of magnetic complexes composed of PEI-Mag2 magnetic nanoparticles, a pCD59-GFPBsd gene construct containing human CD59 gene and DF-Gold as an enhancer, the complexes were associated with the porcine fibroblasts upon application of inhomogeneous magnetic field to promote transgene delivery. After 8-day antibiotic selection, verification of the transgene integration was performed to confirm the generation of transgenic cell lines, which could be further used for somatic cell nuclear transfer

Fig. 2

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

Fig. 3

Magnetic sedimentation of pDNA associated with PEI-Mag2 nanoparticles in the presence of DF-Gold as an enhancer. pDNA associated and magnetically sedimented with PEI-Mag2 nanoparticles in magnetic lipoplexes after incubation at the magnetic plate for 30 min at different enhancer-to-pDNA v/w ratios (4, 3, 2) plotted against magnetic nanoparticle concentrations in terms of iron-to-pDNA w/w ratios (starting pDNA concentration of 2 µg/ml). A range of iron-to-pDNA w/w ratios from 0.0625 to 4 has been examined

Fig. 4

Intracellular localization of magnetic and non-magnetic transfection complexes in PFF cells. Fluorescein-labeled p55pCMV-IVS-luc+ DNA (green) was used to prepare non-magnetic (a lipofection) and magnetic complexes (b magnetofection) at 2:1:4 iron-to-pDNA-to-enhancer (w/w/v) ratio and added to the cells. Following 30 min, 4 h, and 24 h after incubation in a magnetic field, cells were fixed and stained with DAPI (blue), and visualized by confocal microscopy. Arrows indicate the presence of the magnetic lipoplexes in the intracellular compartment. Scale bar 25 µm (Color figure online)

Fig. 5

Interaction of the magnetic transfection complexes with PFF cells. 3D reconstruction of the x,z and y,z slices of the cells exposed to the magnetic transfection complexes containing fluorescently labeled pDNA (green) following 4 h (a) and 24 h (b) after incubation in a magnetic field. Scale bar 10 µm (Color figure online)

Fig. 6

Screening of the pCD59-GFPBsd gene construct. PCR was performed to amplify DNA fragments of 333 and 477 bp. The PCR products were fractionated on 1.5 % agarose gel. Analysis of pCD59-GFPBsd integration with genomic DNA isolated from five transfected PFF cell lines. Lanes 1–5 porcine fetal fibroblasts after magnetofection, lane 6 size marker (Kapa Universal DNA Ladder), lane 7 negative control (porcine DNA), lane 8 negative control (without DNA), lane 9 positive control (pCD59-GFPBsd gene construct)

Fig. 7

Detection of the CD59 mRNA expression in PFF cell lines after magnetofection. PCR amplification followed by agarose gel electrophoresis. a Analysis of pCD59-GFPBsd expression. b Analysis of β-actin expression (cDNA quality control). Lanes 1–5 porcine fetal fibroblasts lines after magnetofection, lane 6 negative control (porcine DNA), lane 7 negative control (without DNA); lane 8 size marker (Kapa Universal DNA Ladder)