Excision efficiency is not strongly coupled to transgenic rate

Cell type-dependent transposition efficiency of sleeping beauty and piggy bac DNA transposons

Orsolya Kolacsek, Zsuzsa Erdei, A. Apáti, Sára Sándor, Zsuzsanna Izsvák, Zoltán Ivics, B. Sarkadi, Tamás I. Orbán

Research output: Contribution to journalArticle

8 Citations (Scopus)

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 because of 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, because of 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 various 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 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.

Original languageEnglish
Pages (from-to)241-252
Number of pages12
JournalHuman gene therapy methods
Volume25
Issue number4
DOIs
Publication statusPublished - Aug 1 2014

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Beauty
DNA Transposable Elements
Transposases
Efficiency
Genetic Therapy
Technology
Transgenes
Real-Time Polymerase Chain Reaction
Research Personnel
Cell Line
Genes
Human Embryonic Stem Cells

ASJC Scopus subject areas

  • Molecular Medicine
  • Applied Microbiology and Biotechnology
  • Genetics(clinical)
  • Genetics
  • Pharmacology
  • Medicine(all)

Cite this

Excision efficiency is not strongly coupled to transgenic rate : Cell type-dependent transposition efficiency of sleeping beauty and piggy bac DNA transposons. / Kolacsek, Orsolya; Erdei, Zsuzsa; Apáti, A.; Sándor, Sára; Izsvák, Zsuzsanna; Ivics, Zoltán; Sarkadi, B.; Orbán, Tamás I.

In: Human gene therapy methods, Vol. 25, No. 4, 01.08.2014, p. 241-252.

Research output: Contribution to journalArticle

Kolacsek, Orsolya ; Erdei, Zsuzsa ; Apáti, A. ; Sándor, Sára ; Izsvák, Zsuzsanna ; Ivics, Zoltán ; Sarkadi, B. ; Orbán, Tamás I. / Excision efficiency is not strongly coupled to transgenic rate : Cell type-dependent transposition efficiency of sleeping beauty and piggy bac DNA transposons. In: Human gene therapy methods. 2014 ; Vol. 25, No. 4. pp. 241-252.
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