gRNA and Cas9 Coexpression
CRISPR-Based Gene Activation
Zebrafish Tol2 Gene Expression Vector
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.
|Genome Biol. 8(Suppl 1): S7 (2007)||Review of Tol2 vectors.|
|Genetics 174: 639 (2006)||Identification of minimal sequences for Tol2 transposable elements.|
|PLoS Genetics 2: e169 (2006)||Large cargo-capacity transposition with a minimal Tol2 transposon.|
Our Tol2 transposon vector system enables efficient insertion of sequences up to 11 kb into the genome of target cells. The Tol2 transposon plasmid along with the helper plasmid are optimized for high copy number replication in E. coli, efficient transfection or electroporation 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 or electroporation 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 or electroporation of 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 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 generally relies on transfection or electroporation. The efficiency of these methods 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 gene of interest is placed here.
Kozak: Kozak consensus sequence. This is placed in front of the start codon of the ORF of interest to facilitate translation initiation in eukaryotes.
ORF: The open reading frame of your gene of interest is placed here.
SV40 late pA: Simian virus 40 late polyadenylation signal. It facilitates transcriptional termination of the upstream ORF.
3' ITR: Tol2 3' 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.
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.