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First published online June 13, 2005
Stem Cells Vol. 23 No. 7 August 2005, pp. 868 -873
doi:10.1634/stemcells.2005-0044; www.StemCells.com
© 2005 AlphaMed Press

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RAPID COMMUNICATIONS

Comparative Analysis of Sequence-Specific DNA Recombination Systems in Human Embryonic Stem Cells

Shen Mynn Tan, Peter Dröge

School of Biological Sciences, Nanyang Technological University, Singapore

Key Words. Site-specific recombination • Human embryonic stem cells • Cre recombinase • {lambda} integrase • Plasmid transfection • {gamma}{delta} resolvase

Correspondence: Peter Dröge, Ph.D., Nanyang Technological University, School of Biological Sciences, 60 Nanyang Drive, 637551, Singapore. Telephone: 65-6316-2809; Fax: 65-6791-3856; e-mail: pdroge{at}ntu.edu.sg

The great potential of human embryonic stem cells (hESCs) in basic research, regenerative medicine, and gene therapy is widely recognized. Controlled manipulation of hESC genomes through sequence-specific DNA recombination (SSR) may play a significant role in future hESC applications. However, very little is known about the functionality of SSR systems in hESCs. We demonstrate here that mutant phage {lambda} integrase, phage P1 Cre recombinase, and mutant {gamma}{delta} resolvase displayed distinct activities on episomal recombination substrates. Interestingly, cofactor-independent {lambda} integrase catalyzed the integrative pathway five times more efficiently than the excisive pathway. Such a degree of directionality in hESCs could be explored for sequential gene insertions into predetermined genomic sequences. We also report an improved, easy-to-use plasmid transfection system that employs silica microspheres and, in combination with SSR, could be applied to hESC genome engineering.




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