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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

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)
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Fig4: 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)

Mentions: In order to evaluate the intracellular localization of magnetic lipoplexes containing fluorescently labeled pDNA, magnetofection was conducted and fluorescent images were taken at 30 min, 4 h, and 24 h after magnetofection. The confocal microscopy confirmed rapid cellular uptake of the complexes during the magnetofection process. As shown in Fig. 4, enhanced uptake of the magnetic transfection complexes into the PFF cells was observed within 30 min after application of the magnetic field. At 4 and 24 h after magnetofection, fluorescent images show that the majority of the magnetic vectors were present around the nucleus (perinuclear localization) and inside the nucleus (Fig. 5 and Supplementary Video S1 and S2). In comparison, cells treated with non-magnetic lipoplexes showed significantly slower and lower uptake. Analysis of pDNA trafficking shows that magnetic vectors accumulate much faster than non-magnetic vectors in the cytoplasm as well as in the nucleus. The images indicate that upon application of the magnetic field, the full-applied magnetic vector dose is sedimented on the cells and internalized within a very short time period and, therefore, the vector dose requirement is considerably reduced. The optical images show the presence of black dots corresponding to the magnetic lipoplexes, which are not observed after lipofection.Fig. 4


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)

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)
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4829627&req=5

Fig4: 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)
Mentions: In order to evaluate the intracellular localization of magnetic lipoplexes containing fluorescently labeled pDNA, magnetofection was conducted and fluorescent images were taken at 30 min, 4 h, and 24 h after magnetofection. The confocal microscopy confirmed rapid cellular uptake of the complexes during the magnetofection process. As shown in Fig. 4, enhanced uptake of the magnetic transfection complexes into the PFF cells was observed within 30 min after application of the magnetic field. At 4 and 24 h after magnetofection, fluorescent images show that the majority of the magnetic vectors were present around the nucleus (perinuclear localization) and inside the nucleus (Fig. 5 and Supplementary Video S1 and S2). In comparison, cells treated with non-magnetic lipoplexes showed significantly slower and lower uptake. Analysis of pDNA trafficking shows that magnetic vectors accumulate much faster than non-magnetic vectors in the cytoplasm as well as in the nucleus. The images indicate that upon application of the magnetic field, the full-applied magnetic vector dose is sedimented on the cells and internalized within a very short time period and, therefore, the vector dose requirement is considerably reduced. The optical images show the presence of black dots corresponding to the magnetic lipoplexes, which are not observed after lipofection.Fig. 4

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