PiggyBac Conditional Gene Knockout Vector (Floxed)

This piggyBac gene knockout vector system utilizes a 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.

The piggyBac system comprises two components: the transposon plasmid and the transposase (helper PBase). The transposon plasmid contains two terminal repeats (TRs) bracketing the region to be transposed. The user-selected promoter and LoxP-flanked gene of interest are cloned into this region. The transposase can be delivered into target cells through two methods. A helper plasmid encoding PBase can be transiently transfected into cells. Alternatively, target cells can be injected with in vitro transcribed PBase mRNA. When the helper PBase and the piggyBac transposon vector are co-introduced into target cells, the transposase produced from the helper would recognize the two TRs on the transposon and insert the flanked region including the two TRs into the host genome. Insertion typically occurs at host chromosomal sites that contain the TTAA sequence, which is duplicated on the two flanks of the integrated fragment. 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.

PiggyBac 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 transposase is reintroduced into the cells, the transposon could get excised from the genome of some cells, footprint-free.

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 piggyBac 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 piggyBac 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.

Very large cargo space: Our transposon vectors can accommodate ~30 kb of total DNA. The plasmid backbone and transposon-related sequences only occupies about 3 kb, leaving plenty of room to accommodate the user's sequence of interest.

Limited cell type range: The delivery of piggyBac vectors into cells relies on transfection. The efficiency of plasmid delivery 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.

piggyBac 5' ITR: 5' inverted terminal repeat. When a DNA sequence is flanked by two ITRs, the piggyBac transpose can recognize them, and insert the flanked region including the two ITRs into the host genome.

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.

rBG pA: Rabbit β-globin 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 signal. It facilitates transcriptional termination of the upstream ORF.

piggyBac 3' ITR: 3' inverted terminal repeat.

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.