PiggyBac/AAV Hybrid Gene Expression Vector

Our piggyBac/AAV hybrid system is highly effective for permanently integrating your gene of interest into the host genome. Delivered via AAV, this system is effective in transducing many mammalian cell types, and almost entirely nonpathogenic in vivo due to its very low immunogenicity. This makes the system ideal for stable and long-term gene expression in various cells and animals.

A piggyBac/AAV hybrid vector is first constructed as a plasmid in E. coli. It is then transfected into packaging cells along with helper plasmids, where the region of the vector between the two inverted terminal repeats (ITRs) is packaged into live AAV virus. Any gene(s) placed in-between the two ITRs, including piggyBac transposon components, are introduced into target cells along with the rest of the viral genome. The single-stranded linear DNA genome is then converted by the host cell DNA polymerase machinery into double-stranded DNA, which forms episomal concatemers in the host cell nucleus.

Next, the transposon flanked by two piggyBac ITRs needs to be mobilized from episomal concatemers in order to integrate into the host genome. Integration requires transient co-expression of a helper transposase, e.g. PBase, in target cells. The transposase recognizes the two ITRs on the transposon and inserts the flanked region including the two ITRs 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. Upon loss of PBase expression, 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.

The tissue tropism (i.e. tissue specificity of infection) of our piggyBac/AAV hybrid vector is decided by its serotype based on different antigenicity of the capsid protein on the viral surface. During cloning, ITRs from AAV2 are used, as this is common practice in the field and does not impact specificity. Packaging helper plasmids include a Rep/Cap plasmid, containing the replication genes from AAV2 and the capsid proteins for a chosen serotype to determine tropism. The table below lists different AAV serotypes and their tissue tropism.

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Our piggyBac/AAV hybrid system along with the helper PBase vector are optimized for high copy number replication in E. coli, high-titer packaging of live virus, efficient transduction both in vitro and in vivo, as well as high-level transgene expression. This system combines the stable integration of the piggyBac system with the high transduction efficiency and versatility of AAV.

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, transduction of mammalian cells with the piggyBac/AAV hybrid vector along with the helper vector can deliver genes carried on the transposon permanently into host cells due to the integration into the host genome.

Safety: AAV is the safest viral vector system available. AAV is inherently replication-deficient, and is not known to cause any human diseases.

Effectiveness in vitro and in vivo: Our vector is often used to transduce cells in live animals, but it can also be used effectively in vitro.

Broad tropism: A wide range of cell and tissue types from commonly used mammalian species such as human, mouse and rat can be readily transduced with our vector when it is packaged into the appropriate AAV serotype. But some cell types may be difficult to transduce, depending on the serotype used (see disadvantages below).

High viral titer: Our vector can be packaged into high titer virus. When AAV virus is obtained through our virus packaging service, titer can reach >10¹³ genome copy per ml (GC/ml).

Small cargo space: AAV has the smallest cargo capacity of any of our viral vector systems. AAV can accommodate a maximum of 4.7 kb of sequence between the ITRs, and due to the transposon components, there is only ~3.7 kb cargo space for user's gene of interest.

Difficulty transducing certain cell types: Our piggyBac/AAV hybrid vector system can transduce many different cell types including non-dividing cells when packaged into the appropriate serotype. However, different AAV serotypes have tropism for different cell types, and certain cell types may be hard to transduce by any serotype.

Technical complexity: The use of this vector system requires the production of live virus in packaging cells followed by the measurement of viral titer. These procedures are technically demanding and time consuming relative to conventional plasmid transfection. These demands can be alleviated by choosing our virus packaging services when ordering your vector.

5' ITR: AAV 5' inverted terminal repeat. In wild type virus, 5' ITR and 3' ITR are essentially identical in sequence. They reside on two ends of the viral genome pointing in opposite directions, where they serve as the origin of viral genome replication.

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

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.

Regulatory element: Allows the user to add the Woodchuck hepatitis virus post-transcriptional regulatory element (WPRE). WPRE enhances virus stability in packaging cells, leading to higher titer of packaged virus; enhances higher expression of transgenes.

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

PiggyBac 3' ITR: PiggyBac 3' inverted terminal repeat. See description for piggyBac 5' ITR.

3' ITR:  AAV 3' inverted terminal repeat. See description for 5' ITR.

Ampicilin: 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.