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First published online June 13, 2005
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2005-0044v1
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Submitted on January 31, 2005
Accepted on May 11, 2005

Rapid Communication

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

Shen Mynn Tan 1 and Peter Dröge 1*

1 School of Biological Sciences, Nanyang Technological University, Singapore

* To whom correspondence should be addressed. E-mail: pdroge{at}ntu.edu.sg.


  Abstract

The great potential of human embryonic stem (hES) cells in basic research, regenerative medicine, and gene therapy is widely recognized. Controlled manipulation of hES cell genomes through sequence-specific DNA recombination (SSR) may play a significant role in future hES cell applications. However, very little is known about the functionality of SSR systems in hES cells. 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, co-factor independent {lambda} integrase catalyzed the integrative pathway five times more efficiently than the excisive pathway. Such a degree of directionality in hES cells could be explored for sequential gene insertions into predetermined genomic sequences. We also report an improved, easy-to-use plasmid transfection system which employs silica microspheres and, in combination with SSR, could be applied for hES cell genome engineering.

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




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