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Using the Cre/ lox system for targeted integration into the human genome: lox FAS‐ lox P pairing and delayed introduction of Cre DNA improve gene swapping efficiency
Author(s) -
Lanza Amanda M.,
Dyess Timothy J.,
Alper Hal S.
Publication year - 2012
Publication title -
biotechnology journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.144
H-Index - 84
eISSN - 1860-7314
pISSN - 1860-6768
DOI - 10.1002/biot.201200034
Subject(s) - cre lox recombination , pairing , genome , computational biology , gene , biology , dna , genetics , physics , transgene , genetically modified mouse , superconductivity , quantum mechanics
Cre recombinase is a commonly‐used genome editing tool suitable for site‐specific integrations in mammalian genomes; however, the efficiency of transgenic swapping events compared to excision remains limited. Here we sought to identify important parameters and limiting factors that influence swapping propensity in this system, especially when using one wild‐type loxP site. To modulate and increase the occurrence of swapping events, we identified two novel parameters. First, we identified the lox FAS‐ loxP pairing, a sequence never before used in mammalian systems, as the best choice for increasing swapping events in human cell lines. Second, for the first time we implicate the importance of delayed introduction of Cre DNA for optimal swapping efficiency. This same modification could potentially be of use to other systems catalyzing trimolecular reactions such as ΦC31 integrase and FLP recombinase where we hypothesize that transport of the exchange cassette is likewise initially rate limiting. The total number of recombination events, but not the ratio of swapping to excision, was found to be influenced by the quantity of Cre DNA transfected. Through this study, we were able to obtain Cre‐mediated swapping frequencies of 8–12% without antibiotic enrichment, which represents nearly an order of magnitude increase over prior reports in the literature.