This piggyBac gene expression vector system utilizes the LoxP-Stop-LoxP (LSL) cassette to achieve Cre-mediated conditional activation of gene expression in mammalian cells and animals. The LSL cassette comprises a LoxP-flanked (aka floxed) triple repeat of the SV40 polyadenylation sequence. The user-selected promoter is placed upstream of the cassette while the user’s gene of interest is placed downstream of it. In the absence of Cre recombinase, the cassette completely blocks transcription of the gene of interest. When Cre is introduced into cells carrying this vector, the cassette is excised, allowing the user-selected promoter to drive the transcription of the gene of interest.
While this vector system can be used in tissue culture cells, it is particular 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 progeny animals carrying both types of transgenes would turn on the gene of interest specifically in cells where the tissue-specific Cre is expressed.
For using this vector system in cell culture, antibiotic or fluorescence based markers can be added to the vector to allow selection or visualization of transfected cells, including the isolation of cells that have permanently integrated the vector in the genome.
This transposon-based, piggyBac 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 user-selected promoter, LSL casette, and the gene of interest are cloned into this region. When the helper and transposon plasmids are both present in target cells, the transposase produced from the helper plasmid recognizes the two TRs on the transposon, and inserts the flanked region 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.
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 and Cre-mediated recombination, please refer to the papers below.
