Open AccessComparative Analysis of Sequence‐Specific DNA Recombination Systems in Human Embryonic Stem Cells
Author(s) -
Tan Shen Mynn,
Dröge Peter
Publication year - 2005
Publication title -
stem cells
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 2.159
H-Index - 229
eISSN - 1549-4918
pISSN - 1066-5099
DOI - 10.1634/stemcells.2005-0044
Subject(s) - biology , integrase , recombinase , cre lox recombination , genetics , site specific recombination , cre recombinase , embryonic stem cell , genome , dna , computational biology , integrases , gene , recombination , transgene , genetically modified mouse
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 λ integrase, phage P1 Cre recombinase, and mutant γδ resolvase displayed distinct activities on episomal recombination substrates. Interestingly, cofactor‐independent λ 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.