Tol2 FLEX Conditional Gene Expression Vector (Cre-Off)

Overview

The Tol2 FLEX conditional Cre-Off gene expression vector combines VectorBuilder’s highly efficient Tol2 vector system with the Cre-responsive FLEX conditional gene expression system to help you achieve transfection-mediated permanent integration of Cre-responsive FLEX switch into the host genome. The FLEX Cre-Off switch utilizes two pairs of LoxP-variant recombination sites flanking a gene of interest in an arrangement which facilitates robust inactivation of gene expression by Cre-dependent inversion of the coding sequence.

The FLEX Cre-Off switch consists of two pairs of heterotypic LoxP-variant recombination sites, namely LoxP having the wild type sequence and Lox2272 having a mutated sequence, flanking an ORF. Both LoxP variants are recognized by Cre, but only identical pairs of LoxP sites can recombine with each other and not with any other variant. The LoxP and Lox2272 sites are organized in an alternating fashion, with an antiparallel orientation for each pair. In the absence of Cre recombinase, the ORF can be expressed under the control of the user-selected promoter. In the presence of Cre, the LoxP and Lox2272 sites undergo recombination with the other LoxP and Lox2272 sites respectively, resulting in the inversion of the ORF to an antisense orientation and excision of one from each pair of identical recombination sites. Inversion of the ORF prevents expression of the gene of interest. Since the ORF is now flanked by two different LoxP-variant sites, no further recombination events will take place even when Cre is present.

The Tol2 vector system is technically simple, utilizing plasmid transfection (rather than viral transduction) to permanently integrate your gene(s) of interest into the host genome. The Tol2 FLEX conditional Cre-Off gene expression vector 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 terminal repeats (TRs) bracketing the region to be transposed. The FLEX Cre-Off switch described above is cloned into this region.

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 FLEX Cre-Off switch 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. Gene expression can then be inactivated in the presence of Cre recombinase upon Cre-mediated inversion of the coding sequence.

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.

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.

References Topic
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
Gene. 216:55 (1998) Characterization of LoxP mutants, including Lox2272
Nat Biotechnol. 21:562 (2003) Development of the FLEX switch system
J Neurosci. 28:7025 (2008) Application of a FLEX switch system

Highlights

The Tol2 FLEX conditional Cre-Off gene expression vector is designed to achieve Cre-mediated conditional inhibition of gene expression in mammalian cells and animals. Expression of the gene of interest is initially under the control of the user-selected promoter and can be permanently silenced by co-expression of Cre recombinase, which will invert the gene of interest to its antisense orientation. 

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.

Advantages

Switch-like gene inactivation: Inversion of the user-selected ORF to its antisense orientation in the presence of Cre recombinase prevents any leaky gene expression.

Stable gene inactivation: Treatment with Cre recombinase will permanently invert the user-selected ORF to its antisense orientation. Upon inversion of the ORF to its antisense orientation followed by excision of one from each pair of similar LoxP sites by recombination, the ORF will be flanked by two different LoxP-variant sites which will prevent further recombination events even when Cre is present. This will permanently prevent transcription of the gene of interest.

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 FLEX conditional Cre-Off gene expression 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.

Disadvantages

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.

Key components

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.

Lox2272: Recombination site for Cre recombinase. Mutated Lox site with two base substitutions of wild type LoxP. Incompatible with LoxP sites. When Cre is present, the LoxP and LoxP2272 sites will be cut and recombine with compatible sites.

LoxP: Recombination site for Cre recombinase. Incompatible with Lox2272 sites. When Cre is present, the LoxP and Lox2272 sites will be cut and recombine with compatible sites.

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, in a sense orientation.

SV40 late pA: Simian virus 40 late polyadenylation signal. It facilitates transcriptional termination of the upstream ORF.

3' ITR: Tol2 3' 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.

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