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Sleeping Beauty transposon-based system for cellular reprogramming and targeted gene insertion in induced pluripotent stem cells

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Item Type:Article
Title:Sleeping Beauty transposon-based system for cellular reprogramming and targeted gene insertion in induced pluripotent stem cells
Creators Name:Grabundzija, I. and Wang, J. and Sebe, A. and Erdei, Z. and Kajdi, R. and Devaraj, A. and Steinemann, D. and Szuhai, K. and Stein, U. and Cantz, T. and Schambach, A. and Baum, C. and Izsvák, Z. and Sarkadi, B. and Ivics, Z.
Abstract:The discovery of direct cell reprogramming and induced pluripotent stem (iPS) cell technology opened up new avenues for the application of non-viral, transposon-based gene delivery systems. The Sleeping Beauty (SB) transposon is highly advanced for versatile genetic manipulations in mammalian cells. We established iPS cell reprogramming of mouse embryonic fibroblasts and human foreskin fibroblasts by transposition of OSKM (Oct4, Sox2, Klf4 and c-Myc) and OSKML (OSKM + Lin28) expression cassettes mobilized by the SB100X hyperactive transposase. The efficiency of iPS cell derivation with SB transposon system was in the range of that obtained with retroviral vectors. Co-expression of the miRNA302/367 cluster together with OSKM significantly improved reprogramming efficiency and accelerated the temporal kinetics of reprogramming. The iPS cells displayed a stable karyotype, and hallmarks of pluripotency including expression of stem cell markers and the ability to differentiate into embryoid bodies in vitro. We demonstrate Cre recombinase-mediated exchange allowing simultaneous removal of the reprogramming cassette and targeted knock-in of an expression cassette of interest into the transposon-tagged locus in mouse iPS cells. This strategy would allow correction of a genetic defect by site-specific insertion of a therapeutic gene construct into 'safe harbor' sites in the genomes of autologous, patient-derived iPS cells.
Keywords:Cell Differentiation, Cultured Cells, DNA Transposable Elements, Gene Knock-In Techniques, Genetic Vectors, HeLa Cells, Induced Pluripotent Stem Cells, Integrases, Nuclear Reprogramming, Transposases, Animals, Mice
Source:Nucleic Acids Research
ISSN:0305-1048
Publisher:Oxford University Press (U.K.)
Volume:41
Number:3
Page Range:1829-1847
Date:1 February 2013
Official Publication:https://doi.org/10.1093/nar/gks1305
PubMed:View item in PubMed

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