Exploring the Cre/Lox System for Targeted Integration Into the Human Genome

Monday, October 17, 2011: 9:10 AM
M100 I (Minneapolis Convention Center)
Amanda M. Lanza and Hal Alper, Chemical Engineering, The University of Texas at Austin, Austin, TX

Efficient site-specific genome editing tools would have great utility in biotechnological and genetics applications.  To this end, Cre recombinase is commonly employed in mammalian transgene editing however the efficiency for recombination mediated cassette exchange is typically below 1% of surviving cells.  One reason for this low efficiency of swapping is the excision function often exhibited by Cre.  Regardless, this efficiency is too low for efficient cell line development applications.  To address this issue, we sought to increase the propensity of Cre recombinase for swapping by undertaking a simultaneous, unbiased evaluation of several parameters found to influence Cre-mediated swapping and excision in human cells (HT1080).  In doing so, we identify parameters that bias total number of recombination events and a separate set of parameters that influence the ratio of swapping to excision events.  In particular, a novel mutant lox site pairing and delayed introduction of Cre are two critical factors for achieving optimal swapping efficiency.  Finally, we create a mathematical model to capture the mechanisms limiting Cre recombinase swapping and highlight two regimes that influence swapping efficiency.  By simultaneously optimizing these parameters, we obtain the highest Cre-mediated swapping frequencies without antibiotic enrichment reported in literature, between 8 and 12%.  These findings indicate the importance of simultaneously examining variables known to influence Cre activity, rather than evaluating these variables in isolation.  Moreover, these results enable more efficient targeted integration into the human genome.

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See more of this Session: Advances In Cell Culture IA: Experimental
See more of this Group/Topical: Food, Pharmaceutical & Bioengineering Division