Sleeping Beauty Conditional Gene Knockout Vector (Floxed)

This sleeping beauty conditional gene knockout vector system utilizes a Cre-mediated system for conditional silencing 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.

Our sleeping beauty conditional gene knockout vector is a highly efficient tool for achieving non-viral, transposon-based delivery of LoxP-flanked gene of interest into target cells. This vector system is derived from the Tc1/mariner superfamily of transposons which were originally isolated from fish genomes and have been transpositionally inactive due to the accumulation of mutations. The sleeping beauty transposon was reconstructed by eliminating such inactivating mutations from sequences of Tc1/mariner transposons found in salmonids.

The sleeping beauty system comprises two components: the transposon plasmid and the transposase (helper). The transposon plasmid contains two inverted/direct repeats (IR/DRs) bracketing the region to be transposed. The transposase can be delivered into target cells through two methods. A helper plasmid encoding transposase can be transiently transfected into cells. Alternatively, target cells can be injected with in vitro transcribed transposase mRNA. When transposon plasmids and the helper are co-introduced into target cells, the transposase produced from the helper would recognize the two IR/DRs on the transposon and insert the flanked region including the two IR/DRs into TA dinucleotide sites of the host genome. At each insertion site, duplicated TA target sites are created, flanking the transposon in the genome. Through both methods of delivering transposase, it is expressed for only a short time. Upon the loss of the helper plasmid or degradation of transposase mRNA, the integration of the transposon into the host genome becomes permanent.

Sleeping Beauty 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). If the sleeping beauty transposase is reintroduced into the cells, the transposon could be excised from the genome of some cells. The excision results in the formation of a “transposon footprint”, consisting of three nucleotides flanked by duplicated TA target sites.

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. Note 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 sleeping beauty transposon-based vector is designed for Cre-mediated conditional gene knockout in mammalian cells and animals. Expression of the gene of interest initially occurs normally, 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.

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 sleeping beauty 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.

Limited cell type range: The delivery of sleeping beauty transposon 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. Additionally, plasmid transfection is largely limited to in vitro applications and rarely used in vivo. These issues limit the use of the sleeping beauty system.

Limited transposon carrying capacity: For transposons between 1.9 and 7.2 Kb, transposition frequency decreases with increase in transposon length.

TATA: TA dinucleotide base-pairs. Increases sleeping beauty transposition efficiency.

IR/DR(L): Inverted/direct repeats of sleeping beauty transposon (Left). Recognized by sleeping beauty transposase; DNA flanked by IR/DR(L) and IR/DR(R) can be transposed by sleeping beauty transposase into TA dinucleotide sites.

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.

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

hPGK promoter: Human phosphoglycerate kinase 1 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.

IR/DR(R): Inverted/direct repeats of sleeping beauty transposon (Right). Recognized by sleeping beauty transposase; DNA flanked by IR/DR(L) and IR/DR(R) can be transposed by sleeping beauty transposase into TA dinucleotide sites.

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.