Our Tol2 vector system is highly effective for inserting foreign DNA into the genome of host cells. This system is technically simple, utilizing plasmid transfection or electroporation to permanently integrate your gene(s) of interest into the host genome.
The system is derived from the Tol2 transposon, which is originally isolated from the teleost fish, medaka (Oryzias latipes). Based on sequence homology, the Tol2 transposon was found to be closely related to the hAT family of non-autonomous elements found throughout vertebrate genomes.
The Tol2 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 inverted terminal repeats (ITRs) bracketing the region to be transposed. The gene to be delivered into host cells is cloned into this region of the transposon plasmid.
When the transposon and helper plasmids are co-transfected or co-electroporated into target cells, the transposase produced from the helper plasmid recognizes the two ITRs on the transposon, and inserts the flanked region 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.
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.
There are two alternative methods for introducing the transposase into target cells. The helper plasmid can be transiently transfected or electroporated into cells, where it will temporarily drive expression of the transposase. Alternatively, target cells can be injected with Tol2 mRNA generated by in vitro transcription from the helper plasmid. In either case, the transposase will only be expressed for a short time. With the loss of the helper plasmid or degradation of transposase mRNA, the integration of the transposon in the host genome becomes permanent. If Tol2 transposase is reintroduced into the cells, the transposon could get excised from the genome of some cells.
For further information about this vector system, please refer to the papers below.
