Regular Plasmid Conditional Gene Knockout Vector (Floxed)

This regular plasmid conditional gene knockout vector is a simple and efficient method to incorporate 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.

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 progeny animals carrying both types of transgenes would knockout the gene of interest, specifically in cells where the tissue-specific Cre is expressed.

For using this vector system in cell culture, antibiotic or fluorescence-based markers can be added to the vector to allow selection or visualization of transfected cells, including the isolation of cells that have permanently integrated the vector in the genome. This vector alone is not sufficient for achieving recombination between pairs of LoxP sites. Coexpression of Cre is required either via a helper vector or mRNA encoding Cre.

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

This vector is designed for Cre-mediated conditional gene knockout in mammalian cells and animals. Expression of the gene of interest initially occurs, but can be permanently silenced by coexpression of Cre recombinase, which will excise the gene of interest.

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

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 for the purpose of Cre-mediated conditional gene knockout.

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 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.

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. It facilitates transcriptional termination of the upstream ORF.

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.