95. Effective Genetic Engineering of Human Embryonic Stem Cells Using the Sleeping Beauty Transposon System

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Human embryonic stem (ES) cells hold great promise for the study of human development and the generation of new therapeutic approaches to tissue regeneration and their genetic modification will play a key role in this development. While viral vectors have been readily used for gene transfer into human ES cells, only limited success has been achieved using non-viral vectors for this purpose. The Sleeping Beauty (SB) transposon system mediates gene insertion into chromosomes, providing a means of durable gene expression in target cells and tissues. To determine whether the SB transposon system can mediate stable gene expression in human ES cells, undifferentiated H9 human ES cells were nucleofected with an SB transposon encoding the firefly luciferase (luc) gene. The vector was designed to be transcriptionally regulated by promoters containing or depleted of CpG dinucleotides, and was delivered with or without a DNA or RNA source of transposase. Transfected human ES cells were maintained in culture, and luc expression was monitored by bioluminescence imaging. Only human ES cells cotransfected with transposon and transposase encoding sequences exhibited luc expression after 2 weeks. Importantly, we found that after 6 weeks, incorporation of a CpG-free promoter increased the average luc activity by 6- to 10-fold compared to a promoter containing CpG sequences. After 5 months, stable expression of luc was observed in human ES cells co-transfected with a transposon containing the CpG-free promoter along with transposase-encoding DNA or RNA. Human ES cells engineered with the CpG-free promoter demonstrated the ability to differentiate into hematopoietic cells in vitro and into teratomas in vivo and maintained luc expression with minimal incidence of epigenetic silencing. We conclude that the SB transposon system provides an effective approach for genetic manipulation of human ES cells and for durable expression using appropriately engineered transposon cargo. Proof-of-principal studies are currently in progress to test gain-of-function and loss-of-function outcomes using the SB system in human ES cells whereby any candidate gene can be evaluated for its role in the differentiation of human ES cells into committed lineages.