The Experts below are selected from a list of 5385 Experts worldwide ranked by ideXlab platform
Jan Vranckx - One of the best experts on this subject based on the ideXlab platform.
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Nonviral transfection strategies for keratinocytes, fibroblasts, and endothelial progenitor cells for ex vivo gene transfer to skin wounds.
Tissue Engineering Part C-methods, 2010Co-Authors: Stijn Dickens, Stefaan Van Den Berge, Benoit Hendrickx, Kristoff Verdonck, Aernout Luttun, Jan VranckxAbstract:In a search for the optimal nonviral gene transfer technique in epidermal and dermal supportive extracellular matrix studies, we investigated the efficiency of late generation liposomal transfection reagents and Nucleofection of fibroblasts (FBs), endothelial progenitor cells (EPCs), and keratinocytes (KCs) as essential indicators of healing skin wounds. FBs, KCs, and EPCs were grown under serum-reduced conditions and manipulated according to optimized in vitro manufacturer protocols. Fugene HD, Effectene, PEI, and Lipofectin were compared to Amaxa Nucleofection. A green fluorescent protein (GFP)-encoded reporter gene plasmid was transfected, and transfection efficiencies were determined by green-fluorescence-activated cell sorting. Normal cell morphologies were observed after either transfection or Nucleofection. For KC cell cultures, Fugene HD resulted in the highest transfection efficiency in human (41%) and porcine (42%) KCs. For EPCs, Effectene was optimal for human-derived cells (42%), whereas nucle...
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Nonviral transfection strategies for keratinocytes, fibroblasts, and endothelial progenitor cells for ex vivo gene transfer to skin wounds.
Tissue engineering. Part C Methods, 2010Co-Authors: Stijn Dickens, Stefaan Van Den Berge, Benoit Hendrickx, Kristoff Verdonck, Aernout Luttun, Jan VranckxAbstract:In a search for the optimal nonviral gene transfer technique in epidermal and dermal supportive extracellular matrix studies, we investigated the efficiency of late generation liposomal transfection reagents and Nucleofection of fibroblasts (FBs), endothelial progenitor cells (EPCs), and keratinocytes (KCs) as essential indicators of healing skin wounds. FBs, KCs, and EPCs were grown under serum-reduced conditions and manipulated according to optimized in vitro manufacturer protocols. Fugene HD, Effectene, PEI, and Lipofectin were compared to Amaxa Nucleofection. A green fluorescent protein (GFP)-encoded reporter gene plasmid was transfected, and transfection efficiencies were determined by green-fluorescence-activated cell sorting. Normal cell morphologies were observed after either transfection or Nucleofection. For KC cell cultures, Fugene HD resulted in the highest transfection efficiency in human (41%) and porcine (42%) KCs. For EPCs, Effectene was optimal for human-derived cells (42%), whereas Nucleofection was optimal (32%) for porcine cells. For FBs, however, Nucleofection resulted in the highest transfection rates in human (46%) and porcine (60%) FBs. For specific epidermal cell studies, Fugene HD was the preferred gene transfer method, whereas Effectene appeared to be the optimal reagent for pro-angiogenic studies. Nucleofection in combination with FBs is the best combination to achieve the highest overall transfection rate and is thus the optimal combination for use in ex vivo gene transfer strategies of wound healing or skin tissue engineering.
Stijn Dickens - One of the best experts on this subject based on the ideXlab platform.
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Nonviral transfection strategies for keratinocytes, fibroblasts, and endothelial progenitor cells for ex vivo gene transfer to skin wounds.
Tissue Engineering Part C-methods, 2010Co-Authors: Stijn Dickens, Stefaan Van Den Berge, Benoit Hendrickx, Kristoff Verdonck, Aernout Luttun, Jan VranckxAbstract:In a search for the optimal nonviral gene transfer technique in epidermal and dermal supportive extracellular matrix studies, we investigated the efficiency of late generation liposomal transfection reagents and Nucleofection of fibroblasts (FBs), endothelial progenitor cells (EPCs), and keratinocytes (KCs) as essential indicators of healing skin wounds. FBs, KCs, and EPCs were grown under serum-reduced conditions and manipulated according to optimized in vitro manufacturer protocols. Fugene HD, Effectene, PEI, and Lipofectin were compared to Amaxa Nucleofection. A green fluorescent protein (GFP)-encoded reporter gene plasmid was transfected, and transfection efficiencies were determined by green-fluorescence-activated cell sorting. Normal cell morphologies were observed after either transfection or Nucleofection. For KC cell cultures, Fugene HD resulted in the highest transfection efficiency in human (41%) and porcine (42%) KCs. For EPCs, Effectene was optimal for human-derived cells (42%), whereas nucle...
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Nonviral transfection strategies for keratinocytes, fibroblasts, and endothelial progenitor cells for ex vivo gene transfer to skin wounds.
Tissue engineering. Part C Methods, 2010Co-Authors: Stijn Dickens, Stefaan Van Den Berge, Benoit Hendrickx, Kristoff Verdonck, Aernout Luttun, Jan VranckxAbstract:In a search for the optimal nonviral gene transfer technique in epidermal and dermal supportive extracellular matrix studies, we investigated the efficiency of late generation liposomal transfection reagents and Nucleofection of fibroblasts (FBs), endothelial progenitor cells (EPCs), and keratinocytes (KCs) as essential indicators of healing skin wounds. FBs, KCs, and EPCs were grown under serum-reduced conditions and manipulated according to optimized in vitro manufacturer protocols. Fugene HD, Effectene, PEI, and Lipofectin were compared to Amaxa Nucleofection. A green fluorescent protein (GFP)-encoded reporter gene plasmid was transfected, and transfection efficiencies were determined by green-fluorescence-activated cell sorting. Normal cell morphologies were observed after either transfection or Nucleofection. For KC cell cultures, Fugene HD resulted in the highest transfection efficiency in human (41%) and porcine (42%) KCs. For EPCs, Effectene was optimal for human-derived cells (42%), whereas Nucleofection was optimal (32%) for porcine cells. For FBs, however, Nucleofection resulted in the highest transfection rates in human (46%) and porcine (60%) FBs. For specific epidermal cell studies, Fugene HD was the preferred gene transfer method, whereas Effectene appeared to be the optimal reagent for pro-angiogenic studies. Nucleofection in combination with FBs is the best combination to achieve the highest overall transfection rate and is thus the optimal combination for use in ex vivo gene transfer strategies of wound healing or skin tissue engineering.
Susan A. Overton - One of the best experts on this subject based on the ideXlab platform.
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non viral transfection of goat germline stem cells by Nucleofection results in production of transgenic sperm after germ cell transplantation
Molecular Reproduction and Development, 2012Co-Authors: Wenxian Zeng, Lin Tang, Alla Bondareva, Jinping Luo, Susan Megee, Mark Modelski, S. Blash, David Melican, Margaret M. Destrempes, Susan A. OvertonAbstract:Germline stem cells (GSCs) can be used for large animal transgenesis, in which GSCs that are genetically manipulated in vitro are transplanted into a recipient testis to generate donor-derived transgenic sperm. The objectives of this study were to explore a non-viral approach for transgene delivery into goat GSCs and to investigate the efficiency of Nucleofection in producing transgenic sperm. Four recipient goats received fractionated irradiation at 8 weeks of age to deplete endogenous GSCs. Germ cell transplantations were performed 8–9 weeks post-irradiation. Donor cells were collected from testes of 9-week-old goats, enriched for GSCs by Staput velocity sedimentation, and transfected by Nucleofection with a transgene construct harboring the human growth hormone gene under the control of the goat beta-casein promoter (GBC) and a chicken beta-globin insulator (CBGI) sequence upstream of the promoter. For each recipient, transfected cells from 10 Nucleofection reactions were pooled, mixed with non-transfected cells to a total of 1.5 × 108 cells in 3 ml, and transplanted into one testis (n = 4 recipients) by ultrasound-guided cannulation of the rete testis. The second testis of each recipient was removed. Semen was collected, starting at 9 months after transplantation, for a period of over a year (a total of 62 ejaculates from four recipients). Nested genomic PCR for hGH and CBGI sequences demonstrated that 31.3% ± 12.6% of ejaculates were positive for both hGH and CBGI. This study provides proof-of-concept that non-viral transfection (Nucleofection) of primary goat germ cells followed by germ cell transplantation results in transgene transmission to sperm in recipient goats. Mol. Reprod. Dev. 79: 255–261, 2012. © 2011 Wiley Periodicals, Inc.
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Non-viral transfection of goat germline stem cells by Nucleofection results in production of transgenic sperm after germ cell transplantation.
Molecular reproduction and development, 2012Co-Authors: Wenxian Zeng, Lin Tang, Alla Bondareva, Jinping Luo, Susan Megee, Mark Modelski, S. Blash, David Melican, Margaret M. Destrempes, Susan A. OvertonAbstract:Germline stem cells (GSCs) can be used for large animal transgenesis, in which GSCs that are genetically manipulated in vitro are transplanted into a recipient testis to generate donor-derived transgenic sperm. The objectives of this study were to explore a non-viral approach for transgene delivery into goat GSCs and to investigate the efficiency of Nucleofection in producing transgenic sperm. Four recipient goats received fractionated irradiation at 8 weeks of age to deplete endogenous GSCs. Germ cell transplantations were performed 8-9 weeks post-irradiation. Donor cells were collected from testes of 9-week-old goats, enriched for GSCs by Staput velocity sedimentation, and transfected by Nucleofection with a transgene construct harboring the human growth hormone gene under the control of the goat beta-casein promoter (GBC) and a chicken beta-globin insulator (CBGI) sequence upstream of the promoter. For each recipient, transfected cells from 10 Nucleofection reactions were pooled, mixed with non-transfected cells to a total of 1.5 × 10(8) cells in 3 ml, and transplanted into one testis (n = 4 recipients) by ultrasound-guided cannulation of the rete testis. The second testis of each recipient was removed. Semen was collected, starting at 9 months after transplantation, for a period of over a year (a total of 62 ejaculates from four recipients). Nested genomic PCR for hGH and CBGI sequences demonstrated that 31.3% ± 12.6% of ejaculates were positive for both hGH and CBGI. This study provides proof-of-concept that non-viral transfection (Nucleofection) of primary goat germ cells followed by germ cell transplantation results in transgene transmission to sperm in recipient goats.
Drew Weissman - One of the best experts on this subject based on the ideXlab platform.
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Nucleofection induces transient eIF2α phosphorylation by GCN2 and PERK
2016Co-Authors: Bart R. Anderson, Katalin Karikó, Drew Weissman, Ph. DAbstract:Nucleofection permits efficient transfection even with difficult cell types such as primary and non-dividing cells, and is used to deliver various nucleic acids including DNA, mRNA, and siRNA. Unlike DNA and siRNA, mRNA is subject to rapid degradation, which necessitates instant early translation following mRNA delivery. We examined factors important in translation following Nucleofection and observed rapid phosphorylation of eukaryotic initiation factor 2 alpha (eIF2α) following Nucleofection, which occurred in the absence of delivered nucleic acid. We studied the involvement of 3 ubiquitous kinases capable of phosphorylating eIF2α in mammalian cells and identified that Nucleofection-mediated phosphorylation of eIF2α was dependent on general control non-derepressible 2 (GCN2) and protein kinase RNA-activated (PKR)-like ER kinase (PERK) but not PKR. A reduction in translation due to eIF2α phosphorylation was observed post Nucleofection demonstrating functional significance. Understanding the impact of Nucleofection on translational machinery has important implications for therapeutics currently under development based on the delivery of mRNA, DNA, and siRNA. Strategies to circumvent eIF2α phosphorylation and other downstream effects of activating GCN2 and PERK will facilitate further advancement of nucleic acid-based therapies
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Nucleofection induces transient eIF2α phosphorylation by GCN2 and PERK.
Gene therapy, 2012Co-Authors: Bart R. Anderson, Katalin Karikó, Drew WeissmanAbstract:Nucleofection permits efficient transfection even with difficult cell types such as primary and non-dividing cells, and is used to deliver various nucleic acids, including DNA, mRNA, and small interfering RNA. Unlike DNA and small interfering RNA, mRNA is subject to rapid degradation, which necessitates instant early translation following mRNA delivery. We examined the factors that are important in translation following Nucleofection and observed rapid phosphorylation of eukaryotic initiation factor 2 alpha (eIF2α) following Nucleofection, which occurred in the absence of the delivered nucleic acid. We studied the involvement of three ubiquitous kinases capable of phosphorylating eIF2α in mammalian cells and identified that Nucleofection-mediated phosphorylation of eIF2α was dependent on general control non-derepressible 2 (GCN2) and RNA-dependent protein kinase (PKR)-like endoplasmic reticulum kinase (PERK) but not PKR. A reduction in translation due to eIF2α phosphorylation was observed post Nucleofection, demonstrating functional significance. Understanding the impact of Nucleofection on translational machinery has important implications for therapeutics currently under development based on the delivery of mRNA, DNA, and small interfering RNA. Strategies to circumvent eIF2α phosphorylation and other downstream effects of activating GCN2 and PERK will facilitate further advancement of nucleic acid-based therapies.
Benoit Hendrickx - One of the best experts on this subject based on the ideXlab platform.
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Nonviral transfection strategies for keratinocytes, fibroblasts, and endothelial progenitor cells for ex vivo gene transfer to skin wounds.
Tissue Engineering Part C-methods, 2010Co-Authors: Stijn Dickens, Stefaan Van Den Berge, Benoit Hendrickx, Kristoff Verdonck, Aernout Luttun, Jan VranckxAbstract:In a search for the optimal nonviral gene transfer technique in epidermal and dermal supportive extracellular matrix studies, we investigated the efficiency of late generation liposomal transfection reagents and Nucleofection of fibroblasts (FBs), endothelial progenitor cells (EPCs), and keratinocytes (KCs) as essential indicators of healing skin wounds. FBs, KCs, and EPCs were grown under serum-reduced conditions and manipulated according to optimized in vitro manufacturer protocols. Fugene HD, Effectene, PEI, and Lipofectin were compared to Amaxa Nucleofection. A green fluorescent protein (GFP)-encoded reporter gene plasmid was transfected, and transfection efficiencies were determined by green-fluorescence-activated cell sorting. Normal cell morphologies were observed after either transfection or Nucleofection. For KC cell cultures, Fugene HD resulted in the highest transfection efficiency in human (41%) and porcine (42%) KCs. For EPCs, Effectene was optimal for human-derived cells (42%), whereas nucle...
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Nonviral transfection strategies for keratinocytes, fibroblasts, and endothelial progenitor cells for ex vivo gene transfer to skin wounds.
Tissue engineering. Part C Methods, 2010Co-Authors: Stijn Dickens, Stefaan Van Den Berge, Benoit Hendrickx, Kristoff Verdonck, Aernout Luttun, Jan VranckxAbstract:In a search for the optimal nonviral gene transfer technique in epidermal and dermal supportive extracellular matrix studies, we investigated the efficiency of late generation liposomal transfection reagents and Nucleofection of fibroblasts (FBs), endothelial progenitor cells (EPCs), and keratinocytes (KCs) as essential indicators of healing skin wounds. FBs, KCs, and EPCs were grown under serum-reduced conditions and manipulated according to optimized in vitro manufacturer protocols. Fugene HD, Effectene, PEI, and Lipofectin were compared to Amaxa Nucleofection. A green fluorescent protein (GFP)-encoded reporter gene plasmid was transfected, and transfection efficiencies were determined by green-fluorescence-activated cell sorting. Normal cell morphologies were observed after either transfection or Nucleofection. For KC cell cultures, Fugene HD resulted in the highest transfection efficiency in human (41%) and porcine (42%) KCs. For EPCs, Effectene was optimal for human-derived cells (42%), whereas Nucleofection was optimal (32%) for porcine cells. For FBs, however, Nucleofection resulted in the highest transfection rates in human (46%) and porcine (60%) FBs. For specific epidermal cell studies, Fugene HD was the preferred gene transfer method, whereas Effectene appeared to be the optimal reagent for pro-angiogenic studies. Nucleofection in combination with FBs is the best combination to achieve the highest overall transfection rate and is thus the optimal combination for use in ex vivo gene transfer strategies of wound healing or skin tissue engineering.
