gRNA and Cas9 Coexpression
CRISPR-Based Gene Activation
Regular Plasmid (FLEX) Conditional Gene Expression Vector (Cre-Off)
This regular plasmid gene expression vector system incorporates a Cre-responsive FLEX switch to achieve Cre-mediated conditional inactivation of gene expression in mammalian cells and animals. This 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.
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.
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 switch system|
This vector is designed for Cre-mediated conditional gene expression in mammalian cells and animals. Expression of the gene of interest is initially under the control of the user-selected promoter, however can be permanently silenced by co-expression of Cre recombinase, which will invert the gene of interest to its antisense orientation.
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.
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 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 a sense orientation.
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.