AAV Inducible Gene Expression Vector (Tet-On)

The AAV inducible gene expression vector combines VectorBuilder’s highly versatile AAV vector system with the Tet-On inducible gene expression system to help you achieve AAV-mediated in vitro and in vivo delivery of tetracycline inducible gene expression cassettes.

The Tet-On inducible gene expression system is a powerful tool to control the timing of expression of the gene(s) of interest (GOI) in mammalian cells. Our Tet-On inducible gene expression vectors are designed to achieve nearly complete silencing of a GOI in the absence of tetracycline and its analogs (e.g. doxycycline), and strong, rapid expression in response to the addition of tetracycline or one of its analogs (e.g. doxycycline). This is achieved through a multicomponent system which incorporates active silencing by the tTS protein in the absence of tetracycline and strong activation by the rtTA protein in the presence of tetracycline. In the absence of tetracycline, the tTS protein derived from the fusion of TetR (Tet repressor protein) and KRAB-AB (the transcriptional repressor domain of Kid-1 protein) binds to the TRE promoter, leading to the active suppression of gene transcription. The rtTA protein, on the other hand, derived from the fusion of a mutant Tet repressor and VP16 (the transcription activator domain of virion protein 16 of herpes simplex virus), binds to the TRE promoter to activate gene transcription only in the presence of tetracycline.

The AAV inducible gene expression 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 virus. The tetracycline inducible gene expression cassette consisting of the tetracycline responsive element (TRE) promoter driving the user-selected GOI and a ubiquitous promoter or tissue-specific promoter driving the regulatory proteins tTS/rtTA is placed in-between the two ITRs, which is introduced into target cells along with the rest of viral genome. Gene expression can then be turned on in the presence of tetracycline.

AAV is effective in transducing many mammalian cell types, and, unlike adenovirus, has very low immunogenicity, being almost entirely nonpathogenic in vivo. This makes the AAV inducible gene expression vector the ideal viral vector system for achieving inducible gene expression in vivo.

The wild-type AAV genome is a linear single-stranded DNA (ssDNA) with two ITRs forming a hairpin structure on each end. It is therefore also known as ssAAV. In order to express genes on ssAAV vectors in host cells, the ssDNA genome needs to first be converted to double-stranded DNA (dsDNA) through two pathways: 1) synthesis of second-strand DNA by the DNA polymerase machinery of host cells using the existing ssDNA genome as the template and the 3' ITR as the priming site; 2) formation of intermolecular dsDNA between the plus- and minus-strand ssAAV genomes. The former pathway is the dominant one.

AAV genomic DNA forms episomal concatemers in the host cell nucleus. In non-dividing cells, these concatemers can remain for the life of the host cells. In dividing cells, AAV DNA is lost through the dilution effect of cell division, because the episomal DNA does not replicate alongside host cell DNA. Random integration of AAV DNA into the host genome can occur but is extremely rare. This is desirable in many gene therapy settings where the potential oncogenic effect of vector integration can pose a significant concern.

A major practical advantage of AAV is that in most cases AAV can be handled in biosafety level 1 (BSL1) facilities. This is due to AAV being inherently replication-deficient, producing little or no inflammation, and causing no known human disease.

Many strains of AAV have been identified in nature. They are divided into different serotypes based on different antigenicity of the capsid protein on the viral surface. Different serotypes can render the virus with different tissue tropism (i.e. tissue specificity of infection). When our AAV vectors are packaged into virus, different serotypes can be conferred to the virus by using different capsid proteins for the packaging. 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.

For further information about this vector system, please refer to the papers below.

Switch-like gene activation: Unlike rtTA only Tet-On systems that usually have significant leaky expression in the absence of induction, our Tet-On gene expression vectors act as true tetracycline-regulated on-and-off switch for controlling gene expression, which can minimize the background expression without induction and result in high sensitivity and high dynamic range of the tetracycline induction.

High-level expression: The TRE promoter can drive very high levels of expression of the GOI in its induced state.

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

Low risk of host genome disruption: Upon transduction into host cells, AAV vectors remain as episomal DNA in the nucleus. The lack of integration into the host genome can be a desirable feature for in vivo human applications, as it reduces the risk of host genome disruption that might lead to cancer.

High viral titer: Our AAV 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).

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 AAV vector when it is packaged into the appropriate serotype. But some cell types may be difficult to transduce, depending on the serotype used (see disadvantages below).

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.

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. In our vector, the components necessary for virus packaging and transduction and for tetracycline-induced gene expression occupies about 2.1 kb, which leaves ~ 2.6 kb cargo space for user's DNA of interest.

Difficulty transducing certain cell types: Our AAV 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 viral vectors 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: 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.

TRE promoter: Tetracycline-responsive element promoter. This element can be regulated by a class of transcription factors (e.g. tTA, rtTA and tTS) whose activities are dependent on tetracycline or its analogs (e.g. doxycycline).

Kozak: Kozak consensus sequence. It is placed in front of the start codon of the ORF of interest 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.

CBh promoter: CMV early enhancer fused to modified chicken β-actin promoter. It drives the ubiquitous expression of the downstream tTS/rtTA cassette.

tTS: Tetracycline-controlled transcriptional silencer. This protein binds to TRE promoter to actively suppress gene transcription only in the absence of tetracycline and its analogs (e.g. doxycycline).

T2A: Self-cleaving 2A peptide from thosea asigna virus that allows multiple proteins to be made from a polycistronic transcript containing multiple ORFs separated by T2A. The cleavage occurs through a putative “ribosomal skipping” mechanism.

rtTA: Reverse tetracycline responsive transcriptional activator M2. This protein binds to TRE promoter to activate gene transcription only in the presence of tetracycline or its analogs (e.g. doxycycline). It has higher sensitivity to the inducing drug and lower leaky activity in the absence of the drug compared to its predecessor.

BGH pA: Bovine growth hormone polyadenylation. It facilitates transcriptional termination of the upstream tTS/rtTA cassette.

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.