gRNA and Cas9 Coexpression
CRISPR-Based Gene Activation
Regular Plasmid (FLEX) Conditional Gene Expression Vector (Cre-Switch)
This regular plasmid gene expression vector system incorporates a Cre-responsive FLEX switch to achieve permanent Cre-mediated switching between the expression of two ORFs in mammalian cells and animals. This FLEX Cre-switch system utilizes two pairs of LoxP-variant recombination sites flanking two antiparallel ORFs in an arrangement which facilitates activation of one gene while repressing the other by Cre-dependent inversion of both ORFs.
The FLEX Cre-switch system consists of two pairs of heterotypic LoxP-variant recombination sites, namely LoxP, having the wild type sequence and Lox2272, having a mutated sequence flanking a pair of ORFs. 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 two ORFs are in an opposite orientation with respect to one-another, such that one ORF is in its proper sense orientation, while the other is in an antisense orientation. The LoxP and Lox2272 sites are organized in an alternating fashion, with an antiparallel orientation for each pair. In the absence of Cre recombinase, while the first ORF is expressed under the control of the user-selected promoter, the second ORF is not expressed due to its antisense orientation. 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 both ORFs and excision of one from each pair of identical recombination sites. Inversion of the ORFs results in silencing of the first ORF (which will now be in an antisense orientation) and allows expression of the second ORF (which will now be in a sense orientation).
While this vector system can be used in tissue culture cells, it is particularly suitable for the generation of transgenic animals. Transgenic animals carrying such a vector originally express the first user-selected ORF, however when crossed to an animal carrying a tissue-specific Cre transgene, expression of the second user-selected ORF will be activated while silencing the first ORF in the progeny animals carrying both types of transgenes, specifically in cells where the tissue-specific Cre is expressed and the user-selected promoter is active.
Antibiotic or fluorescence-based markers can be added to this vector to allow selection or visualization of transfected cells, including the isolation of cells that have permanently integrated the vector in their genome.
For further information about this vector system, please refer to the papers below.
|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 system|
This vector is designed for Cre-mediated switching between expression of two ORFs in mammalian cells and animals. Expression is under the control of a user-selected promoter, and can be permanently switched from one user-selected ORF to another by co-expression of Cre recombinase.
Switch-like regulation: Opposite orientation of the two ORFs ensures that while the ORF in the sense orientation is expressed, the ORF in the antisense orientation is repressed without any leaky gene expression.
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 vector can accommodate ~30 kb of total DNA. The plasmid backbone only occupies about 3 kb, leaving plenty of room to accommodate the user's sequence of interest.
High-level expression: Conventional transfection of plasmids can often result in very high copy numbers in cells (up to several thousand copies per cell). This can lead to very high expression levels of the genes carried on the vector.
Suitability for in vivo applications: While this vector system can be used in tissue culture cells, it is particularly suitable for the generation of transgenic animals with Cre-mediated conditional gene expression.
Non-integration of vector DNA: When used in cell culture, plasmid DNA generally integrates into the host genome at only a very low frequency (one per 102 to 106 cells depending on cell type). Drug resistance or fluorescence markers incorporated into the plasmid can be used to isolate cells stably integrating the plasmid by drug selection or cell sorting after extended culture.
Limited cell type range: The efficiency of plasmid delivery in cell culture can vary greatly from cell type to cell type, and often requires optimization. Primary cells are often harder to transfect than immortalized cell lines, and some cell types are notoriously difficult to transfect.
Non-uniformity of gene delivery: Although a successful transfection can result in very high average copy number of the transfected plasmid vector per cell, this can be highly non-uniform. Some cells can carry many copies while others carry very few or none. This is unlike transduction by virus-based vectors which tends to result in relatively uniform gene delivery into cells.
Promoter: The promoter driving your genes 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 #1: The open reading frame of a gene of interest is placed here, in a sense orientation. This gene can be expressed without Cre-mediated recombination.
ORF #2: The open reading frame of a gene of interest is placed here, in an antisense orientation. This gene can only be expressed after Cre-mediated recombination.
SV40 late pA: Simian virus 40 late 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.
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.