Efficient non-viral integration and stable gene expression in multipotent adult progenitor cells

Andrew Wilber, Fernando Ulloa Montoya, Luke Hammer, Branden S Moriarity, Aron M. Geurts, David A Largaespada, Catherine M. Verfaillie, R S Mc Ivor, Uma Lakshmipathy

Research output: Contribution to journalArticle

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Abstract

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.

Original languageEnglish (US)
Article number717069
JournalStem Cells International
DOIs
StatePublished - Dec 1 2011

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Transposases
Beauty
Stem Cells
Gene Expression
Clone Cells
Kanamycin Kinase
Transgenic Rats
Integrases
Aptitude
Genetic Engineering
Southern Blotting
Bacteriophages
Endothelium
Cell Biology
Plasmids
Fibroblasts
Liver
Genes

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Efficient non-viral integration and stable gene expression in multipotent adult progenitor cells. / Wilber, Andrew; Ulloa Montoya, Fernando; Hammer, Luke; Moriarity, Branden S; Geurts, Aron M.; Largaespada, David A; Verfaillie, Catherine M.; Mc Ivor, R S; Lakshmipathy, Uma.

In: Stem Cells International, 01.12.2011.

Research output: Contribution to journalArticle

Wilber, Andrew ; Ulloa Montoya, Fernando ; Hammer, Luke ; Moriarity, Branden S ; Geurts, Aron M. ; Largaespada, David A ; Verfaillie, Catherine M. ; Mc Ivor, R S ; Lakshmipathy, Uma. / Efficient non-viral integration and stable gene expression in multipotent adult progenitor cells. In: Stem Cells International. 2011.
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AU - Largaespada, David A

AU - Verfaillie, Catherine M.

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