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Excision efficiency is not strongly coupled to transgenic rate: cell type dependent transposition efficiency of Sleeping Beauty and piggyBac DNA transposons

Item Type:Article
Title:Excision efficiency is not strongly coupled to transgenic rate: cell type dependent transposition efficiency of Sleeping Beauty and piggyBac DNA transposons
Creators Name:Kolacsek, O. and Erdei, Z. and Apáti, A. and Sándor, S. and Izsvák, Z. and Ivics, Z. and Sarkadi, B. and Orbán, T.I.
Abstract:The Sleeping Beauty (SB) and piggyBac (PB) DNA transposons represent an emerging new gene delivery technology, potentially suitable for human gene therapy applications. Previous studies pointed to important differences between these transposon systems, depending on the cell types examined and the methodologies applied. However, efficiencies cannot always be compared due to differences in applications. In addition, "overproduction inhibition", a phenomenon believed to be a characteristic of DNA transposons, can remarkably reduce the overall transgenic rate, emphasizing the importance of transposase dose applied. Therefore, due to lack of comprehensive analysis, researchers are forced to optimize the technology for their own "in house" platforms. In this study, we investigated the transposition of several SB (SB11, SB32, SB100X) and PB (mPB and hyPB) variants in different cell types at three levels: comparing the excision efficiency of the reaction by real-time PCR, testing the overall transgenic rate by detecting cells with stable integrations, and determining the average copy number when using different transposon systems and conditions. We concluded that a high excision activity is not always followed by a higher transgenic rate, as exemplified by the hyperactive transposases, indicating that the excision and the integration steps of transposition are not strongly coupled as previously thought. In general, all levels of transposition show remarkable differences depending on the transposase used and cell lines examined, being the least efficient in human embryonic stem cells (hESCs). In spite of the comparably low activity in those special cell types, the hyperactive SB100X and hyPB systems could be used in hESCs with similar transgenic efficiency and with reasonably low (2-3) transgene copy numbers, indicating their potential applicability for gene therapy purposes in the future.
Keywords:Sleeping Beauty, PiggyBac, Transposon, Excision Efficiency, Transgenic Rate, Overproduction Inhibition, Embryonic Stem Cells
Source:Human Gene Therapy Methods
ISSN:1946-6536
Publisher:Mary Ann Liebert (U.S.A.)
Volume:25
Number:4
Page Range:241-252
Date:August 2014
Official Publication:https://doi.org/10.1089/hgtb.2013.149
External Fulltext:View full text on PubMed Central
PubMed:View item in PubMed

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