Single-Stranded AAV (ssAAV)
Tet Regulatory Protein Expression Vector
VectorBuilder’s single-stranded adeno-associated virus (ssAAV) Tet regulatory protein expression vector can be used for AAV-based delivery of Tet regulatory proteins (tTS, rtTA, etc.) into mammalian cells to help you achieve tetracycline-inducible expression of target genes placed downstream of a tetracycline responsive-element (TRE) promoter.
The Tet-On inducible 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 ssAAV Tet regulatory protein expression vector is first constructed as a plasmid in E. coli. The Tet regulatory protein expression cassette consisting of the Tet regulatory protein(s) driven a user-selected promoter is placed in between the two inverted terminal repeats (ITRs). It is then transfected into packaging cells along with helper plasmids, where the Tet regulatory protein expression cassette between the two inverted terminal repeats (ITRs) is packaged into active virus. Any gene(s) placed in-between the two ITRs are introduced into target cells along with the rest of viral genome.
While our ssAAV Tet regulatory protein expression vector includes an expression cassette consisting of the Tet regulatory protein(s) driven by a user-selected promoter, the GOI driven by the TRE promoter must be provided using a separate helper vector to achieve tetracycline induced gene expression in the presence of tetracycline, while minimizing leaky expression in the absence 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. The lack of integration 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. The table below lists different AAV serotypes and their tissue tropism.
List by Serotype
List by Tissue Type
||Smooth muscle, skeletal muscle, CNS, brain, lung, retina, inner ear, pancreas, heart, liver
||Smooth muscle, CNS, brain, liver, pancreas, kidney, retina, inner ear, testes
||Smooth muscle, liver, lung
||CNS, retina, lung, kidney, heart
||Smooth muscle, CNS, brain, lung, retina, heart
||Smooth muscle, heart, lung, pancreas, adipose, liver
||Lung, liver, inner ear
||Smooth muscle, retina, CNS, brain, liver
||Smooth muscle, CNS, brain, retina, inner ear, liver, pancreas, heart, kidney, adipose
||Smooth muscle, skeletal muscle, lung, liver, heart, pancreas, CNS, retina, inner ear, testes, kidney, adipose
||Smooth muscle, lung, liver, heart, pancreas, CNS, retina, kidney
||Liver, heart, kidney, spleen
||Liver, brain, spleen, kidney
||Endothelial cell, retina
||Retina, inner ear
||Recommended AAV serotypes
||AAV1, AAV2, AAV3, AAV5, AAV6, AAV7, AAV8, AAV9, AAVrh10
||AAV1, AAV2, AAV4, AAV5, AAV7, AAV8, AAV9, AAVrh10, AAV-PHP.eB
||AAV1, AAV2, AAV5, AAV7, AAV8, AAV-DJ/8
||AAV1, AAV2, AAV4, AAV5, AAV7, AAV8, AAV9, AAVrh10, AAV2-QuadYF, AAV2.7m8
||AAV1, AAV2, AAV6.2, AAV8, AAV9, AAV2.7m8
||AAV1, AAV3, AAV4, AAV5, AAV6, AAV6.2, AAV9, AAVrh10
||AAV1, AAV2, AAV3, AAV6, AAV6.2, AAV7, AAV8, AAV9, AAVrh10, AAV-DJ, AAV-DJ/8
||AAV1, AAV2, AAV6, AAV8, AAV9, AAVrh10
||AAV1, AAV4, AAV5, AAV6, AAV8, AAV9, AAVrh10, AAV-DJ
||AAV2, AAV4, AAV8, AAV9, AAVrh10, AAV-DJ, AAV-DJ/8
||AAV6, AAV8, AAV9
For further information about this vector system, please refer to the papers below.