Excision Efficiency is Not Strongly Coupled to Transgenic Rate: Cell Type-dependent Transposition Efficiency of Sleeping Beauty and PiggyBac DNA Transposons

Overview

Journal Hum Gene Ther Methods

Publisher Mary Ann Liebert

Specialties Genetics
Pharmacology

Date 2014 Jul 22

PMID 25045962

Citations 13

Authors

Orsolya Kolacsek

Zsuzsa Erdei

Agota Apati

Sara Sandor

Zsuzsanna Izsvak

Zoltan Ivics

Balazs Sarkadi

Tamas I Orban

Affiliations

Soon will be listed here.

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.

Citing Articles

Partial Disturbance of Microprocessor Function in Human Stem Cells Carrying a Heterozygous Mutation in the DGCR8 Gene.

Ree D, Fothi A, Varga N, Kolacsek O, Orban T, Apati A Genes (Basel). 2022; 13(11).

PMID: 36360162 PMC: 9689658. DOI: 10.3390/genes13111925.

Microglia modulate proliferation, neurite generation and differentiation of human neural progenitor cells.

Lilienberg J, Apati A, Rethelyi J, Homolya L Front Cell Dev Biol. 2022; 10:997028.

PMID: 36313581 PMC: 9606406. DOI: 10.3389/fcell.2022.997028.

Functional Characterization of the N-Terminal Disordered Region of the Transposase.

Wachtl G, Schad E, Huszar K, Palazzo A, Ivics Z, Tantos A Int J Mol Sci. 2022; 23(18).

PMID: 36142241 PMC: 9499001. DOI: 10.3390/ijms231810317.

Progress of Transposon Vector System for Production of Recombinant Therapeutic Proteins in Mammalian Cells.

Wei M, Mi C, Jing C, Wang T Front Bioeng Biotechnol. 2022; 10:879222.

PMID: 35600890 PMC: 9114503. DOI: 10.3389/fbioe.2022.879222.

Pharmacological Modulation of Neurite Outgrowth in Human Neural Progenitor Cells by Inhibiting Non-muscle Myosin II.

Lilienberg J, Hegyi Z, Szabo E, Hathy E, Malnasi-Csizmadia A, Rethelyi J Front Cell Dev Biol. 2021; 9:719636.

PMID: 34604221 PMC: 8484915. DOI: 10.3389/fcell.2021.719636.

References

Pledger D, Coates C . Mutant Mos1 mariner transposons are hyperactive in Aedes aegypti. Insect Biochem Mol Biol. 2005; 35(10):1199-207. DOI: 10.1016/j.ibmb.2005.06.002. View

Singer T, McConnell M, Marchetto M, Coufal N, Gage F . LINE-1 retrotransposons: mediators of somatic variation in neuronal genomes?. Trends Neurosci. 2010; 33(8):345-54. PMC: 2916067. DOI: 10.1016/j.tins.2010.04.001. View

Ivics Z, Hackett P, Plasterk R, Izsvak Z . Molecular reconstruction of Sleeping Beauty, a Tc1-like transposon from fish, and its transposition in human cells. Cell. 1997; 91(4):501-10. DOI: 10.1016/s0092-8674(00)80436-5. View

Zayed H, Izsvak Z, Khare D, Heinemann U, Ivics Z . The DNA-bending protein HMGB1 is a cellular cofactor of Sleeping Beauty transposition. Nucleic Acids Res. 2003; 31(9):2313-22. PMC: 154227. DOI: 10.1093/nar/gkg341. View

Walisko O, Izsvak Z, Szabo K, Kaufman C, Herold S, Ivics Z . Sleeping Beauty transposase modulates cell-cycle progression through interaction with Miz-1. Proc Natl Acad Sci U S A. 2006; 103(11):4062-7. PMC: 1449646. DOI: 10.1073/pnas.0507683103. View

Doherty J, Huye L, Yusa K, Zhou L, Craig N, Wilson M . Hyperactive piggyBac gene transfer in human cells and in vivo. Hum Gene Ther. 2011; 23(3):311-20. PMC: 3300075. DOI: 10.1089/hum.2011.138. View

Mates L, Chuah M, Belay E, Jerchow B, Manoj N, Acosta-Sanchez A . Molecular evolution of a novel hyperactive Sleeping Beauty transposase enables robust stable gene transfer in vertebrates. Nat Genet. 2009; 41(6):753-61. DOI: 10.1038/ng.343. View

Burnight E, Staber J, Korsakov P, Li X, Brett B, Scheetz T . A Hyperactive Transposase Promotes Persistent Gene Transfer of a piggyBac DNA Transposon. Mol Ther Nucleic Acids. 2013; 1:e50. PMC: 3499692. DOI: 10.1038/mtna.2012.12. View

Sarkadi B, Orban T, Szakacs G, Varady G, Schamberger A, Erdei Z . Evaluation of ABCG2 expression in human embryonic stem cells: crossing the same river twice?. Stem Cells. 2009; 28(1):174-6. DOI: 10.1002/stem.262. View

10.

Woltjen K, Michael I, Mohseni P, Desai R, Mileikovsky M, Hamalainen R . piggyBac transposition reprograms fibroblasts to induced pluripotent stem cells. Nature. 2009; 458(7239):766-70. PMC: 3758996. DOI: 10.1038/nature07863. View

11.

Sharma N, Hollensen A, Bak R, Staunstrup N, Schroder L, Mikkelsen J . The impact of cHS4 insulators on DNA transposon vector mobilization and silencing in retinal pigment epithelium cells. PLoS One. 2012; 7(10):e48421. PMC: 3482222. DOI: 10.1371/journal.pone.0048421. View

12.

Wilson M, Coates C, George Jr A . PiggyBac transposon-mediated gene transfer in human cells. Mol Ther. 2006; 15(1):139-45. DOI: 10.1038/sj.mt.6300028. View

13.

Liang Q, Kong J, Stalker J, Bradley A . Chromosomal mobilization and reintegration of Sleeping Beauty and PiggyBac transposons. Genesis. 2009; 47(6):404-8. DOI: 10.1002/dvg.20508. View

14.

Cai Y, Bak R, Krogh L, Staunstrup N, Moldt B, Corydon T . DNA transposition by protein transduction of the piggyBac transposase from lentiviral Gag precursors. Nucleic Acids Res. 2013; 42(4):e28. PMC: 3936723. DOI: 10.1093/nar/gkt1163. View

15.

Li M, Pettitt S, Eckert S, Ning Z, Rice S, Cadinanos J . The piggyBac transposon displays local and distant reintegration preferences and can cause mutations at noncanonical integration sites. Mol Cell Biol. 2013; 33(7):1317-30. PMC: 3624274. DOI: 10.1128/MCB.00670-12. View

16.

Cadinanos J, Bradley A . Generation of an inducible and optimized piggyBac transposon system. Nucleic Acids Res. 2007; 35(12):e87. PMC: 1919496. DOI: 10.1093/nar/gkm446. View

17.

Goodier J, Kazazian Jr H . Retrotransposons revisited: the restraint and rehabilitation of parasites. Cell. 2008; 135(1):23-35. DOI: 10.1016/j.cell.2008.09.022. View

18.

Liu G, Aronovich E, Cui Z, Whitley C, Hackett P . Excision of Sleeping Beauty transposons: parameters and applications to gene therapy. J Gene Med. 2004; 6(5):574-83. PMC: 1865527. DOI: 10.1002/jgm.486. View

19.

Balciunas D, Wangensteen K, Wilber A, Bell J, Geurts A, Sivasubbu S . Harnessing a high cargo-capacity transposon for genetic applications in vertebrates. PLoS Genet. 2006; 2(11):e169. PMC: 1635535. DOI: 10.1371/journal.pgen.0020169. View

20.

Galvan D, Nakazawa Y, Kaja A, Kettlun C, Cooper L, Rooney C . Genome-wide mapping of PiggyBac transposon integrations in primary human T cells. J Immunother. 2009; 32(8):837-44. PMC: 2796288. DOI: 10.1097/CJI.0b013e3181b2914c. View