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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.
|Cell. 157:77 (2014)||Review on non-coding RNAs|
|Front Genet. 6:2 (2015)||Review on functionality of non-coding RNAs|
|Genome Biol. 8(Suppl 1): S7 (2007)||Review of Tol2 vectors.|
|Genetics 174: 639-649 (2006)||Identification of minimal sequences for Tol2 transposable elements.|
|PLoS Genetics 2: e169 (2006)||Large cargo-capacity transposition with a minimal Tol2 transposon.|
The Tol2 non-coding RNA expression vector along with the helper plasmid are optimized for high copy number replication in E. coli, efficient transfection into a wide range of target cells, and high-level expression of the transgene carried on the vector.
Permanent integration of vector DNA: Conventional transfection results in almost entirely transient delivery of DNA into host cells due to the loss of DNA over time. This problem is especially prominent in rapidly dividing cells. In contrast, transfection of mammalian cells with the Tol2 transposon plasmid along with the helper plasmid (or introduction of Tol2 mRNA) can deliver genes carried on the transposon permanently into host cells due to the integration of the transposon into the host genome.
Technical simplicity: Delivering plasmid vectors into cells by conventional transfection is technically straightforward, and far easier than virus-based vectors which require the packaging of live virus.
Very large cargo space: Our Tol2 transposon vector can accommodate ~11 kb of total DNA. The plasmid backbone and transposon-related sequences only occupies about 3 kb, leaving plenty of room to accommodate the user's sequence of interest.
Limited cell type range: The delivery of Tol2 vectors into cells relies on transfection. The efficiency of transfection can vary greatly from cell type to cell type. Non-dividing cells are often more difficult to transfect than dividing cells, and primary cells are often harder to transfect than immortalized cell lines. Some important cell types, such as neurons and pancreatic β cells, are notoriously difficult to transfect. These issues limit the use of the Tol2 system.
5' ITR: Tol2 5' terminal repeat. When a DNA sequence is flanked by two ITRs, the Tol2 transposase can recognize them, and insert the flanked region including the two ITRs into the host genome.
Promoter: The promoter driving your non-coding RNA of interest is placed here.
Non-coding RNA: The non-coding RNA of your interest is placed here.
SV40 late pA: Simian virus 40 late polyadenylation signal. It facilitates transcriptional termination of the upstream non-coding RNA.
3' ITR: 3' inverted terminal repeat.
Ampicillin: Ampicillin resistance gene. It allows the plasmid to be maintained by ampicillin selection in E. coli.
pUC ori: pUC origin of replication. Plasmids carrying this origin exist in high copy numbers in E. coli.