Finally, when the two LoxP sites are placed on two different DNA molecules, recombination of intermolecular LoxP sites can lead to site-specific insertion or reciprocal translocation between the two DNA. On the other hand when the two LoxP sites are placed in the reciprocal orientation, Cre recombinase can inverse the sequence between the two LoxP sites. When the two LoxP sites are arranged in same orientation, the Cre recombinase can excise any intervening sequence flanked by LoxP sites.
Outcomes of the Cre-mediated recombination are determined by the orientations of two LoxP sites. In this genetic binary system, Cre recombinase catalyzes site-specific recombination between two LoxP sites, which are 34 bp DNA fragment containing an asymmetric 8 bp (base pair) spacer flanked by two 13 bp inverted repeats. The Cre-LoxP system, derived from P1 bacteriophage, is a site-specific recombination system extensively used in experimental organisms. 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. We also demonstrate that the recombination between Lox2272 sites is much less efficient than wild type LoxP sites.
Thus this method expends current molecular biology toolbox. This protocol is simple and highly efficient. We have presented an in vitro protocol to invert the ORF in FLEX based plasmids.
These assays revealed, significant impairment in recombination between Lox2272 sites as compared wild type LoxP sites in the FLEX plasmids. To understand this, we performed in vitro recombination assays. To our surprise, single step recombination efficiency between the two mutant Lox2272 sites turned out very low. This protocol, requiring as little as 30 min and 50 ng of plasmid, has a cloning efficiency of 40–50%. 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. Therefore developing a universal protocol for this purpose is useful as it could save a lot of valuable time and lab resources. 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. These plasmids are designed to only work in combination with Cre. Since its first publication, numerous FLEX switch plasmids have been generated. 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.
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