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An accumulative site‐specific gene integration system using cre recombinase‐mediated cassette exchange
Author(s) -
Kameyama Yujiro,
Kawabe Yoshinori,
Ito Akira,
Kamihira Masamichi
Publication year - 2010
Publication title -
biotechnology and bioengineering
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.136
H-Index - 189
eISSN - 1097-0290
pISSN - 0006-3592
DOI - 10.1002/bit.22619
Subject(s) - cre recombinase , recombinase , site specific recombination , chinese hamster ovary cell , cre lox recombination , plasmid , gene , biology , gene targeting , flp frt recombination , genetics , genome , computational biology , transgene , recombination , genetic recombination , cell culture , genetically modified mouse
Abstract The Cre‐ loxP system is frequently used for site‐specific recombination in animal cells. The equilibrium and specificity of the recombination reaction can be controlled using mutated loxP s. In the present study, we designed an accumulative site‐specific gene integration system using Cre recombinase and mutated loxP s in which the Cre‐mediated cassette exchange reaction is infinitely repeatable for target gene integration into loxP target sites. To evaluate the feasibility and usefulness of this system, a series of integration reactions were repeated and confirmed in vitro using Cre recombinase protein and plasmids. Accumulative gene integration was also performed on the genome of Chinese hamster ovary (CHO) cells. The results indicated that the system was applicable for repeated gene integration of multiple genes to the target sites on both plasmids and CHO cell genomes. This gene integration system provides a novel strategy for gene amplification and for biological analyses of gene function through the genetic modification of cells and organisms. Biotechnol. Bioeng. 2010;105: 1106–1114. © 2009 Wiley Periodicals, Inc.