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Stem Cells InternationalVolume 2011 2011, Article ID 717069, 14 pages

Research Article

Center for Genome Engineering, Gene Therapy Program, Institute of Human Genetics, University of Minnesota, Minneapolis, MN 55455, USA

Department of Genetics, Cell Biology and Development, University of Minnesota, Minneapolis, MN 55455, USA

Department of Medical Microbiology, Immunology and Cell Biology and Simmons Cancer Institute, Southern Illinois University School of Medicine, Springfield, IL 62702, USA

Department of Medicine, Stem Cell Institute, University of Minnesota, Minneapolis, MN 55455, USA

Department of Physiology, Human and Molecular Genetics Center, Medical College of Wisconsin, Milwaukee, WI 53226, USA

Interdepartmental Stem Cell Institute, Katholieke University Leuven, Leuven, Belgium

Life Technologies, 5781 Van Allen Way, Carlsbad, CA 92008, USA

Received 14 January 2011; Revised 1 June 2011; Accepted 15 June 2011

Academic Editor: Mark G. Carter

Copyright © 2011 Andrew Wilber et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

Non-viral integrating systems, PhiC31 phage integrase C31, 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 C31 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 C31. Sequence identification of chromosomal junction sites indicated a random profile for SB-mediated integrants and a more restricted profile for C31 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 C31 are effective non-viral integrating systems for genetic engineering of MAPC in basic studies of stem cell biology.





Autor: Andrew Wilber, Fernando Ulloa Montoya, Luke Hammer, Branden S. Moriarity, Aron M. Geurts, David A. Largaespada, Catherine M.

Fuente: https://www.hindawi.com/



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