Sleeping Beauty Gene Expression Vector

Overview

Our sleeping beauty vector system is highly effective for inserting foreign DNA into the genome of host cells. This system is technically simple, utilizing plasmid transfection (rather than viral transduction) to permanently integrate your gene(s) of interest into the host genome.

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 development of this synthetic transposon has provided a highly efficient transposon-based method for achieving transgenesis and insertional mutagenesis in vertebrates.

The sleeping beauty 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 inverted/direct repeats IR/DR(R) bracketing the region to be transposed. The gene to be delivered into host cells 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 IR/DR(R)s on the transposon, and insert the flanked region including the two IR/DR(R)s into TA dinucleotide sites of the host genome.

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.) 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
Cell. 91:501 (1997) Molecular reconstruction of the sleeping beauty transposon
Mol Ther. 8:108 (2003) Gene transfer into genome of human cells by sleeping beauty transposon
Mol Ther. 9:147 (2004) Review on biology & applications of sleeping beauty transposon

Highlights

Our sleeping beauty transposon plasmid along with the helper plasmid 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

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.

Disadvantages

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.

Key components

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 driving your gene of interest is placed here.

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.

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.

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

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.