Tol2 Conditional Gene Knockout Vector (Floxed)

The Tol2 conditional gene knockout vector system combines VectorBuilder’s highly efficient Tol2 vector system with the Cre-responsive gene knockout system to help you achieve transfection-mediated permanent integration of Cre-mediated conditional inactivation of gene expression in mammalian cells and animals. This floxed system comprises LoxP recombination sites flanking a gene of interest to facilitate inactivation of gene expression by Cre-dependent deletion of the coding sequence. In the absence of Cre recombinase, expression of the gene of interest is permitted. When Cre is introduced into cells carrying this vector, the gene of interest is permanently excised.

The Tol2 vector system is technically simple, utilizing transfection (rather than viral transduction) to permanently integrate your gene(s) of interest into the host genome. The Tol2 system comprises two components: the transposon plasmid and the transposase. The transposon plasmid contains two inverted terminal repeats (ITRs) bracketing the region to be transposed. The LoxP-flanked gene of interest described above is cloned into this region. After delivery of a Tol2 transposase, the region between the ITRs of the transposon is integrated into the host genome. Gene expression can then be inactivated in the presence of Cre recombinase upon Cre-mediated inversion of the coding sequence. 

The transposase can be delivered into target cells through two methods. The helper plasmid can be transiently transfected into cells. Alternatively, target cells can be injected with transposase mRNA generated by in vitro transcription from the helper 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 insert the flanked region including the two ITRs into the host genome. 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. Insertion occurs without any significant bias for the insertion site sequence. This is unlike transposon systems which have specific target consensus sites. For example, piggyBac transposons are typically inserted at sites containing the sequence TTAA. Through both methods of delivering transposase, it is expressed for only a short time. Upon the loss of the helper plasmid or degradation of transposase mRNA, the integration of the transposon into the host genome becomes permanent. 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.) If Tol2 transposase is reintroduced into the cells, the transposon could get excised from the genome of some cells, resulting in either precise or imprecise excisions with indels created.

While this vector system can be used in tissue culture cells, it is particularly 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 gene of interest would be turned off in the progeny animals carrying both types of transgenes, specifically in cells where the tissue-specific Cre is expressed and the user-selected promoter driving the gene of interest is active.

For further information about this vector system and Cre-mediated recombination, please refer to the papers below.

This Tol2 conditional gene knockout vector is designed to achieve Cre-mediated conditional gene deletion in mammalian cells and animals. Expression of the gene of interest initially occurs normally, but can be permanently silenced by coexpression of Cre recombinase, which will excise the gene of interest. This vector along with the helper plasmid encoding the Tol2 transposase 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.

Stable gene inactivation: Treatment with Cre recombinase will permanently remove the sequence encoding the gene of interest and prevents its transcription.

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 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 conditional gene knockout vector can accommodate ~11 kb of total DNA. The plasmid backbone, transposon-related sequences and the sequences necessary for Cre-mediated recombination only occupy about 3.1 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 that will drive expression of your gene of interest is placed here.

LoxP: Recombination site for Cre recombinase. When Cre is present the region flanked by the two LoxP sites will be excised.

Kozak: Kozak consensus sequence. It is placed in front of the start codon of the ORF of interest because it is believed 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.

hPGK promoter: Human phosphoglycerate kinase 1 promoter.  It drives the ubiquitous expression of the downstream marker gene.

Marker: A drug selection gene (such as neomycin resistance), a visually detectable gene (such as EGFP), or a dual-reporter gene (such as EGFP/Neo). This allows cells transduced with the vector to be selected and/or visualized.

3' ITR: Tol2 3' terminal repeat.

pUC ori: pUC origin of replication. Plasmids carrying this origin exist in high copy numbers in E. coli.

Ampicillin: Ampicillin resistance gene. It allows the plasmid to be maintained by ampicillin selection in E. coli.