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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

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
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Fig1: 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

Mentions: In this study, magnetofection technology was used to deliver a pCD59-GFPBsd gene construct containing a gene encoding a protein involved in the regulation of the human immune response system, into porcine fetal fibroblasts (PFFs) (Fig. 1). These cells may be a source of nuclei in the SCNT method to generate transgenic animals for the purpose of xenotransplantation. PEI-Mag2 iron oxide nanoparticles with magnetic core and appropriate coating [18] were used to formulate efficient magnetic pDNA lipoplexes. To visualize the rapid internalization of magnetic complexes, images of fluorescently labeled pDNA were taken. To confirm integration of the transgene into the genome and its expression, PCR and RT-PCR analysis after 8-day selection was performed.Fig. 1


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)

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
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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

Fig1: 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
Mentions: In this study, magnetofection technology was used to deliver a pCD59-GFPBsd gene construct containing a gene encoding a protein involved in the regulation of the human immune response system, into porcine fetal fibroblasts (PFFs) (Fig. 1). These cells may be a source of nuclei in the SCNT method to generate transgenic animals for the purpose of xenotransplantation. PEI-Mag2 iron oxide nanoparticles with magnetic core and appropriate coating [18] were used to formulate efficient magnetic pDNA lipoplexes. To visualize the rapid internalization of magnetic complexes, images of fluorescently labeled pDNA were taken. To confirm integration of the transgene into the genome and its expression, PCR and RT-PCR analysis after 8-day selection was performed.Fig. 1

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