PiggyBac FLEX Conditional Gene Expression Vector (Cre-On)

Overview

The piggyBac FLEX conditional Cre-On gene expression vector combines VectorBuilder’s highly efficient piggyBac 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-On switch utilizes two pairs of LoxP-variant recombination sites flanking a gene of interest in an arrangement which completely inhibits gene expression in the absence of Cre and activates gene expression upon Cre-dependent inversion of the coding sequence.

The FLEX Cre-On 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 which is in the reverse (antisense) orientation relative to the promoter. 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 is not expressed due to its antisense orientation relative to the 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 a sense orientation and excision of one from each pair of identical recombination sites. This allows the user-selected promoter to drive the transcription 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 piggyBac 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 piggyBac FLEX conditional Cre-On gene expression 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-On switch described above is cloned into this region.

When the helper and transposon plasmids are co-transfected into target cells, the transposase produced from the helper would recognize the two TRs on the transposon and insert the flanked FLEX Cre-On switch including the two TRs into the host genome. Insertion typically occurs at host chromosomal sites that contain the TTAA sequence, which is duplicated on the two flanks of the integrated fragment. Gene expression can then be activated in the presence of Cre 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 system is crossed to an animal carrying a tissue-specific Cre transgene, the gene of interest would be turned on 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.

PiggyBac 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.) Because the helper plasmid is only transiently transfected into host cells, it will get lost over time. With the loss of the helper plasmid, the integration of the transposon in the genome of host cells becomes permanent. If these cells are transfected with the helper plasmid again, the transposon could get excised from the genome of some cells, footprint free.

For further information about this vector system, please refer to the papers below.

References Topic
Mol Cell Biochem. 354:301 (2011) Review on the piggyBac system
Cell. 122:473 (2005) Efficient transposition of the piggyBac (PB) transposon in mammalian cells and mice
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 piggyBac FLEX conditional Cre-On gene expression vector is designed to achieve Cre-mediated conditional gene expression in mammalian cells and animals. Expression of the gene of interest is initially silent, however can be permanently activated by co-expression of Cre recombinase, which will invert the gene of interest to its coding orientation. In the presence of Cre, expression of the gene of interest is under the control of the user-selected promoter.

This vector along with the helper plasmid encoding the piggyBac 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 activation: Antisense orientation of the user-selected ORF, prevents any leaky gene expression prior to Cre-mediated recombination. Other conditional gene expression systems, including LoxP-Stop-LoxP can have some low-level leaky (read-through) expression under certain circumstances.

Stable gene activation: Treatment with Cre recombinase will permanently invert the user-selected ORF to its sense orientation. Upon inversion of the ORF to its sense 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 allow transcription of the gene of interest, driven by the promoter chosen by the user.

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 piggyBac 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 piggyBac FLEX conditional Cre-On gene expression vector can accommodate ~30 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 piggyBac 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 piggyBac system.

Key components

5' ITR: 5' inverted terminal repeat. When a DNA sequence is flanked by two ITRs, the piggyBac transpose 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.

ORF: The open reading frame of your gene of interest is placed here, in an antisense orientation.

Kozak: Kozak consensus sequence. It is placed in front of the start codon of the ORF of interest to facilitate translation initiation in eukaryotes.

rBG pA:  Rabbit beta-globin polyadenylation signal. It facilitates transcriptional termination of the upstream ORF.

CMV promoter: Human cytomegalovirus immediate early 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.

BGH pA: Bovine growth hormone polyadenylation signal. It facilitates transcriptional termination of the upstream ORF.

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.

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