The piggyBac FLEX conditional Cre-Off gene expression vector combines VectorBuilder’s highly efficient piggyBac 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-Off switch utilizes two pairs of LoxP-variant recombination sites flanking a gene of interest in an arrangement which facilitates robust inactivation of gene expression by Cre-dependent inversion of the coding sequence.
The FLEX Cre-Off 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. 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 can be expressed under the control of the user-selected 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 an antisense orientation and excision of one from each pair of identical recombination sites. Inversion of the ORF prevents expression 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 piggyBac 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 piggyBac FLEX conditional Cre-Off gene expression vector 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-Off switch described above is cloned into this region.
When the helper and transposon plasmids are co-transfected into target cells, the transposase produced from the helper would recognize the two TRs on the transposon and insert the flanked FLEX Cre-Off switch including the two TRs into the host genome. Insertion typically occurs at host chromosomal sites that contain the TTAA sequence, which is duplicated on the two flanks of the integrated fragment. Gene expression can then be inactivated in the presence of Cre recombinase 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 is crossed to an animal carrying a tissue-specific Cre transgene, the gene of interest would be turned off 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.
PiggyBac 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.) Because the helper plasmid is only transiently transfected into host cells, it will get lost over time. With the loss of the helper plasmid, the integration of the transposon in the genome of host cells becomes permanent. If these cells are transfected with the helper plasmid again, the transposon could get excised from the genome of some cells, footprint free.
For further information about this vector system, please refer to the papers below.