Efficient Non-Viral Integration and Stable Gene Expression in Multipotent Adult Progenitor Cells
Non-viral integrating systems, PhiC31 phage integrase (ϕC31), and Sleeping Beauty transposase (SB), provide an effective method for ex vivo gene delivery into cells. Here, we used a plasmid-encoding GFP and neomycin phosphotransferase along with recognition sequences for both ϕC31 and SB integratin...
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| Main Authors: | , , , , , , , , |
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| Format: | Article |
| Language: | English |
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Wiley
2011-01-01
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| Series: | Stem Cells International |
| Online Access: | http://dx.doi.org/10.4061/2011/717069 |
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| _version_ | 1850167058358075392 |
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| author | Andrew Wilber Fernando Ulloa Montoya Luke Hammer Branden S. Moriarity Aron M. Geurts David A. Largaespada Catherine M. Verfaillie R. Scott McIvor Uma Lakshmipathy |
| author_facet | Andrew Wilber Fernando Ulloa Montoya Luke Hammer Branden S. Moriarity Aron M. Geurts David A. Largaespada Catherine M. Verfaillie R. Scott McIvor Uma Lakshmipathy |
| author_sort | Andrew Wilber |
| collection | DOAJ |
| description | Non-viral integrating systems, PhiC31 phage integrase (ϕC31), and Sleeping Beauty transposase (SB), provide an effective method for ex vivo gene delivery into cells. Here, we used a plasmid-encoding GFP and neomycin phosphotransferase along with recognition sequences for both ϕC31 and SB integrating systems to demonstrate that both systems effectively mediated integration in cultured human fibroblasts and in rat multipotent adult progenitor cells (rMAPC). Southern blot analysis of G418-resistant rMAPC clones showed a 2-fold higher number of SB-mediated insertions per clone compared to ϕC31. Sequence identification of chromosomal junction sites indicated a random profile for SB-mediated integrants and a more restricted profile for ϕC31 integrants. Transgenic rMAPC generated with both systems maintained their ability to differentiate into liver and endothelium albeit with marked attenuation of GFP expression. We conclude that both SB and ϕC31 are effective non-viral integrating systems for genetic engineering of MAPC in basic studies of stem cell biology. |
| format | Article |
| id | doaj-art-db48085b129a49e08c0edd2a02e48100 |
| institution | OA Journals |
| issn | 1687-966X 1687-9678 |
| language | English |
| publishDate | 2011-01-01 |
| publisher | Wiley |
| record_format | Article |
| series | Stem Cells International |
| spelling | doaj-art-db48085b129a49e08c0edd2a02e481002025-08-20T02:21:17ZengWileyStem Cells International1687-966X1687-96782011-01-01201110.4061/2011/717069717069Efficient Non-Viral Integration and Stable Gene Expression in Multipotent Adult Progenitor CellsAndrew Wilber0Fernando Ulloa Montoya1Luke Hammer2Branden S. Moriarity3Aron M. Geurts4David A. Largaespada5Catherine M. Verfaillie6R. Scott McIvor7Uma Lakshmipathy8Center for Genome Engineering, Gene Therapy Program, Institute of Human Genetics, University of Minnesota, Minneapolis, MN 55455, USADepartment of Medicine, Stem Cell Institute, University of Minnesota, Minneapolis, MN 55455, USADepartment of Medicine, Stem Cell Institute, University of Minnesota, Minneapolis, MN 55455, USACenter for Genome Engineering, Gene Therapy Program, Institute of Human Genetics, University of Minnesota, Minneapolis, MN 55455, USACenter for Genome Engineering, Gene Therapy Program, Institute of Human Genetics, University of Minnesota, Minneapolis, MN 55455, USACenter for Genome Engineering, Gene Therapy Program, Institute of Human Genetics, University of Minnesota, Minneapolis, MN 55455, USADepartment of Medicine, Stem Cell Institute, University of Minnesota, Minneapolis, MN 55455, USACenter for Genome Engineering, Gene Therapy Program, Institute of Human Genetics, University of Minnesota, Minneapolis, MN 55455, USADepartment of Medicine, Stem Cell Institute, University of Minnesota, Minneapolis, MN 55455, USANon-viral integrating systems, PhiC31 phage integrase (ϕC31), and Sleeping Beauty transposase (SB), provide an effective method for ex vivo gene delivery into cells. Here, we used a plasmid-encoding GFP and neomycin phosphotransferase along with recognition sequences for both ϕC31 and SB integrating systems to demonstrate that both systems effectively mediated integration in cultured human fibroblasts and in rat multipotent adult progenitor cells (rMAPC). Southern blot analysis of G418-resistant rMAPC clones showed a 2-fold higher number of SB-mediated insertions per clone compared to ϕC31. Sequence identification of chromosomal junction sites indicated a random profile for SB-mediated integrants and a more restricted profile for ϕC31 integrants. Transgenic rMAPC generated with both systems maintained their ability to differentiate into liver and endothelium albeit with marked attenuation of GFP expression. We conclude that both SB and ϕC31 are effective non-viral integrating systems for genetic engineering of MAPC in basic studies of stem cell biology.http://dx.doi.org/10.4061/2011/717069 |
| spellingShingle | Andrew Wilber Fernando Ulloa Montoya Luke Hammer Branden S. Moriarity Aron M. Geurts David A. Largaespada Catherine M. Verfaillie R. Scott McIvor Uma Lakshmipathy Efficient Non-Viral Integration and Stable Gene Expression in Multipotent Adult Progenitor Cells Stem Cells International |
| title | Efficient Non-Viral Integration and Stable Gene Expression in Multipotent Adult Progenitor Cells |
| title_full | Efficient Non-Viral Integration and Stable Gene Expression in Multipotent Adult Progenitor Cells |
| title_fullStr | Efficient Non-Viral Integration and Stable Gene Expression in Multipotent Adult Progenitor Cells |
| title_full_unstemmed | Efficient Non-Viral Integration and Stable Gene Expression in Multipotent Adult Progenitor Cells |
| title_short | Efficient Non-Viral Integration and Stable Gene Expression in Multipotent Adult Progenitor Cells |
| title_sort | efficient non viral integration and stable gene expression in multipotent adult progenitor cells |
| url | http://dx.doi.org/10.4061/2011/717069 |
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