Establishing and Validation of Modified CRE/Loxp System Useful In Temporal and Spatial Control of Genetic Knocking Out Using In Vitro Approaches by Induced Alpha Complementation

DNA recombinases are used to manipulate the genomic structure and to control the genetic expression in all living organisms. Cre is a P1 bacteriophage-derived tyrosine recombinase mediating the site-specific recombination between two loxP DNA recognition sites. Cre/loxP system allows generation of tissue specific mutations and is widely used in bioengineering and in mouse genetics. However, various factors limit its applicability such as lack of temporal control over its recombination activity, presence of cellular compensatory mechanisms, loss of site-specificity at high expression levels and its limited use for conditional recombination in certain brain structures due to a lack of sufficiently selective promoters. One approach used to overcome these drawbacks is the so-called split-iCRE technique that employs complementation of split-iCRE fragments via artificial FKBP12-rapamycin or ɑ-helix interactions. Rapamycin is a pharmacologically active substance while leucine zipper-mediated complementation so far only insufficiently restores recombinase activity. Here we introduce a system for controlling ɑ-complementation of two independent iCRE fragments under the control of two different promoters to reconstitute recombinase activity. Unsplit-iCRE was split between Lys130 and Asp132. Fragments were complemented restoring ~ 95% of the recombinase activity with very little background activity. Adding an external nuclear localization signal to the C-terminal fragment resulted in even higher enzymatic activity. Using an extended rigid linker between the polypeptide and the yeast GCN4-coil/coil leucine zipper domains was more efficient than a semi-flexible separator. The system was validated by knocking out the essential circadian clock component Bmal1 (Arntl) in MEF cells resulting in a loss of clock function in MEF cells.