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PiggyBac Toolbox

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Mobile Genetic Elements

Part of the book series: Methods in Molecular Biology ((MIMB,volume 859))

Abstract

The PiggyBac (PB) transposon system was originally derived from the cabbage looper moth Trichoplusia ni and represents one of the most promising transposon systems to date. Engineering of the PB transposase enzyme (PBase) and its cognate transposon DNA elements resulted in a substantial increase in transposition activities. Consequently, this has greatly enhanced the versatility of the PB toolbox. It is now widely used for stable gene delivery into a broad variety of cell types from different species, including mammalian cells. This opened up new perspectives for potential therapeutic applications in the fields of gene therapy and regenerative medicine. In particular, we have recently demonstrated that PB transposons could be used to stably deliver genes into human CD34+ hematopoietic stem cells (HSCs) resulting in sustained transgene expression in its differentiated progeny. The PB transposon system is particularly attractive for the generation of induced pluripotent stem cells (iPS). Typically, this can be accomplished by stable gene transfer of genes encoding one or more reprogramming factors (i.e., c-MYC, KLF-4, OCT-4, and/or SOX-2). We have generated a PB-based nonviral reprogramming toolbox that contains different combinations of these reprogramming genes. The main advantage of using this PB toolbox for iPS generation is that the reprogramming cassette can be excised by de novo transposase expression, without leaving any molecular trace in the target cell genome. This “traceless excision” paradigm obviates potential risks associated with inadvertent re-expression of reprogramming factors in the iPS progeny. These various applications in gene therapy, stem cell engineering, and regenerative medicine underscore the emerging versatility of the PB toolbox.

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Abbreviations

BSA:

Bovine serum albumin

CAG:

CMV early enhancer/chicken b-actin promoter

CFU-E:

Erythroid colony forming unit

CFU-GM:

Granulocyte/monocyte/macrophage colony forming unit

ePB:

Enhanced PB system (14)

FACS:

Fluorescence-activated Cell Sorter

GFP:

Green fluorescence protein

hFlt3-L:

Human FMS-like tyrosine kinase 3-ligand

hIL-3:

Human interleukin-3

hIL-6:

Human interleukin-6

HSCs:

Hematopoietic stem cells

hSCF:

Human stem cell factor

hTPO:

Human thrombopoietin

hyPB:

Hyperactive PB (10)

iPS:

Induced pluripotent stem cells

IRs:

Inverted terminal repeats

mPB:

Codon-optimized mouse PB (19)

PB:

PiggyBac

PBase:

PB transposase enzyme

PBaseCO-MT :

Human or mouse codon-optimized PB transposase (Chuah-VandenDriessche lab, unpublished)

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Acknowledgments

This work was supported by the 7th EU framework programme (grant agreement no 222878, PERSIST) FWO, GOA EPIGEN (VUB), EHA, and AFM.

Author information

Authors and Affiliations

  1. Division of Gene Therapy & Regenerative Medicine, Free University of Brussels (VUB), University Medical Center – Jette, Brussels, Belgium

    Mario Di Matteo, Janka Mátrai, Tewodros Firdissa, Thierry VandenDriessche & Marinee K. L. Chuah

  2. Department of Molecular Cardiovascular Medicine, University of Leuven, University Hospital Campus, Gasthuisberg, Belgium

    Mario Di Matteo, Janka Mátrai, Tewodros Firdissa, Thierry VandenDriessche & Marinee K. L. Chuah

  3. Division of Gene Therapy & Regenerative Medicine, ree University of Brussels (VUB), Faculty of Medicine & Pharmacy, University Medical Center – Jette, Laarbeeklaan 103, B-1090, Brussels, Belgium

    Eyayu Belay

Authors
  1. Mario Di Matteo
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  2. Janka Mátrai
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  3. Eyayu Belay
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  4. Tewodros Firdissa
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  5. Thierry VandenDriessche
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  6. Marinee K. L. Chuah
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Corresponding author

Correspondence to Marinee K. L. Chuah .

Editor information

Editors and Affiliations

  1. , Physiologie de la Reproduction, UMR INRA-CNRS 6175, n/a, Nouzilly Cedex, 37380, France

    Yves Bigot

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Di Matteo, M., Mátrai, J., Belay, E., Firdissa, T., VandenDriessche, T., Chuah, M.K.L. (2012). PiggyBac Toolbox. In: Bigot, Y. (eds) Mobile Genetic Elements. Methods in Molecular Biology, vol 859. Humana Press. https://doi.org/10.1007/978-1-61779-603-6_14

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  • DOI: https://doi.org/10.1007/978-1-61779-603-6_14

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  • Publisher Name: Humana Press

  • Print ISBN: 978-1-61779-602-9

  • Online ISBN: 978-1-61779-603-6

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