The Tol2 FLEX conditional Cre-On gene expression vector combines VectorBuilder’s highly efficient Tol2 vector system with the Cre-responsive FLEX conditional gene expression system to help you achieve transfection-mediated permanent integration of Cre-responsive FLEX switch into the host genome. The 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.
The Tol2 vector system is technically simple, utilizing plasmid transfection (rather than viral transduction) to permanently integrate your gene(s) of interest into the host genome. The Tol2 FLEX conditional Cre-On gene expression system contains two vectors, both engineered as E. coli plasmids. One vector, referred to as the helper plasmid, encodes the transposase. The other vector, referred to as the transposon plasmid, contains two terminal repeats (TRs) bracketing the region to be transposed. The FLEX Cre-On switch described above is cloned into this region.
When the transposon and helper plasmids are co-transfected into target cells, the transposase produced from the helper plasmid would recognize the two ITRs on the transposon and insert the flanked FLEX Cre-On switch including the two ITRs into the host genome. Insertion occurs without any significant bias with respect to insertion site sequence. This is unlike transposon systems which have specific target consensus sites. For example, piggyBac transposons typically inserts at sites containing the sequence TTAA. Gene expression can then be activated in the presence of Cre upon Cre-mediated inversion of the coding sequence.
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 system 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.
Tol2 is a class II transposon, meaning that it moves in a cut-and-paste manner, hopping from place to place without leaving copies behind. (In contrast, class I transposons move in a copy-and-paste manner.) Tol2 integrates as a single copy through a cut-and-paste mechanism. At each insertion site, the Tol2 transposase creates an 8 bp duplication, resulting in identical 8 bp direct repeats flanking each transposon integration site in the genome.
For further information about this vector system, please refer to the papers below.