Regular Plasmid FLEX Conditional Gene Expression Vector (Cre-On)
This regular plasmid gene expression vector system incorporates a Cre-responsive FLEX switch to achieve Cre-mediated conditional activation of gene expression in mammalian cells and animals. This FLEX Cre-On switch utilizes two pairs of LoxP-variant recombination sites flanking a gene of interest in an arrangement which completely inhibits gene expression in the absence of Cre, and activates gene expression upon Cre-dependent inversion of the coding sequence.
The FLEX Cre-On switch consists of two pairs of heterotypic LoxP-variant recombination sites, namely LoxP, having the wild type sequence and Lox2272, having a mutated sequence, flanking an ORF which is in the reverse (antisense) orientation relative to the promoter. Both LoxP variants are recognized by Cre, but only identical pairs of LoxP sites can recombine with each other and not with any other variant. The LoxP and Lox2272 sites are organized in an alternating fashion, with an antiparallel orientation for each pair. In the absence of Cre recombinase, the ORF is not expressed due to its antisense orientation relative to the promoter. In the presence of Cre, the LoxP and Lox2272 sites undergo recombination with the other LoxP and Lox2272 sites respectively, resulting in the inversion of the ORF to a sense orientation and excision of one from each pair of identical recombination sites. This allows the user-selected promoter to drive the transcription of the gene of interest. Since the ORF is now flanked by two different LoxP-variant sites, no further recombination events will take place even when Cre is present.
While this vector system can be used in tissue culture cells, it is particularly suitable for the generation of transgenic animals. When a transgenic animal carrying such a vector is crossed to an animal carrying a tissue-specific Cre transgene, the gene of interest would be turned on in the progeny animals carrying both types of transgenes, specifically in cells where the tissue-specific Cre is expressed and the user-selected promoter driving the gene of interest is active.
Antibiotic or fluorescence-based markers can be added to this vector to allow selection or visualization of transfected cells, including the isolation of cells that have permanently integrated the vector in their genome.
For further information about this vector system, please refer to the papers below.