A rapid in vitro method to flip back the double-floxed inverted open reading frame in a plasmid.

Background: The LoxP site based genetic switch, the FLEX, also known as DIO (Double-Floxed Inverted Open reading frame), was invented to turn on gene expression via Cre-mediated recombination. Since its first publication, numerous FLEX switch plasmids have been generated. These plasmids are designed to only work in combination with Cre. However on many occasions it is necessary to covert these FLEX plasmids back into constitutive expression plasmids so that they can also be used in non-Cre-expressing cells and in non-genetically modified animal models. Therefore developing a universal protocol for this purpose is useful as it could save a lot of valuable time and lab resources.

Result: Here we report a simple, quick, and cost-efficient protocol to invert the orientation of the open reading frame (ORF) within FLEX switch containing plasmids using commercial Cre recombinase. This protocol, requiring as little as 30 min and 50 ng of plasmid, has a cloning efficiency of 40-50%. To our surprise, single step recombination efficiency between the two mutant Lox2272 sites turned out very low. To understand this, we performed in vitro recombination assays. These assays revealed, significant impairment in recombination between Lox2272 sites as compared wild type LoxP sites in the FLEX plasmids.

Conclusion: We have presented an in vitro protocol to invert the ORF in FLEX based plasmids. This protocol is simple and highly efficient. Thus this method expends current molecular biology toolbox. We also demonstrate that the recombination between Lox2272 sites is much less efficient than wild type LoxP sites. This result has important implication for evaluating the efficacy of FLEX switch in biological systems and provides a rationale for future development of higher efficiency LoxP mutants.