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First published online August 2, 2007
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2007-0026v1
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Submitted on January 10, 2007
Accepted on July 25, 2007

TECHNOLOGY DEVELOPMENT

Efficient and Stable Transgene Expression in Human Embryonic Stem Cells Using Transposon-Mediated Gene Transfer

Andrew Wilber 1, Jonathan L. Linehan 2, Xinghui Tian 2, Petter S. Woll 2, Julie K. Morris 2, Lalitha R. Belur 1, R. Scott McIvor 1, Dan S. Kaufman 2*

1 The Arnold and Mabel Beckman Center for Transposon Research; Gene Therapy Program; Institute of Human Genetics; and Department of Genetics, Cell Biology and Development, University of Minnesota, Minneapolis, Minnesota 55455
2 Department of Medicine, Stem Cell Institute, University of Minnesota, Minneapolis, Minnesota 55455

* To whom correspondence should be addressed. E-mail: kaufm020{at}umn.edu.


  Abstract

Efficient and stable genetic modification of human embryonic stem (ES) cells is required to realize the full scientific and potential therapeutic use of these cells. Currently, only limited success toward this goal has been achieved without using a viral vector. The Sleeping Beauty (SB) transposon system mediates non-viral gene insertion and stable expression in target cells and tissues. Here, we demonstrate use of the non-viral Sleeping Beauty (SB) transposon system to effectively mediate stable gene transfer in human ES cells. Transposons encoding (i) GFP coupled to the zeocin gene or (ii) the firefly luciferase (luc) gene were effectively delivered to undifferentiated human ES cells with either a DNA or RNA source of transposase. Only human ES cells co-transfected with transposon and transposase-encoding sequences exhibited transgene expression after one week in culture. Molecular analysis of transposon integrants indicated that 98% of stable gene transfer resulted from transposition. Stable luc expression was observed up to five months in human ES cells co-transfected with a transposon along with either DNA or RNA encoding SB transposase. Genetically engineered human ES cells demonstrated the ability to differentiate into teratomas in vivo and mature hematopoietic cells in vitro while maintaining stable transgene expression. We conclude that the SB transposon system provides an effective approach with several advantages for genetic manipulation and durable gene expression in human ES cells.

Key Words. Human Embryonic Stem Cells, Sleeping Beauty, Non-viral integration, Stable Expression, Bioluminescence Imaging




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[Abstract] [Full Text] [PDF]


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