The Tol2 non-coding RNA expression vector is a highly effective tool for transfection-based permanent integration of non-coding RNAs into the host genome of mammalian cells. Non-coding RNAs include a wide variety of short (<30 nucleotides) and long (>200 nucleotides) functional RNA molecules such as micro RNAs (miRNAs), small interfering RNAs (siRNAs), piwi-interacting RNAs (piRNAs), small nuclear RNAs (snRNAs), small nucleolar RNAs (snoRNAs), large intergenic non-coding RNAs (lincRNAs), intronic long non-coding RNAs (intronic lncRNAs), natural antisense transcripts (NATs), enhancer RNAs (eRNAs) and promoter-associated RNAs (PARs), none of which are translated into proteins, however have been found to play important roles in many cellular processes such as DNA replication, epigenetic regulation, transcriptional and post-transcriptional regulation and translation regulation.
The Tol2 non-coding RNA expression vector uses an RNA polymerase II promoter to drive the expression of the user-selected non-coding RNA gene. This allows the use of tissue-specific, inducible, or variable-strength promoters, enabling a variety of experimental applications. For RNA polymerase II-mediated transcription, the start site is typically in the 3' region of the promoter while the termination site is within the polyA signal sequence. As a result, the transcript generated from this vector does not correspond precisely to the selected non-coding RNA gene, but contains some additional sequences both upstream and downstream.
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 non-coding RNA of interest to be delivered into host cells along with a user-selected promoter is cloned into this region of the transposon plasmid.
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 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.
For further information about this vector system, please refer to the papers below.
