AAV (FLEX) Conditional Gene Expression Vector (Cre-Switch)
The AAV (FLEX) conditional Cre-Switch gene expression vector combines VectorBuilder’s highly versatile AAV vector system with the Cre-responsive (FLEX) conditional gene expression system to help you achieve AAV-mediated in vitro and in vivo delivery of FLEX Cre-Switch for Cre-induced switching between the expression of two ORFs. The FLEX Cre-Switch system utilizes two pairs of LoxP-variant recombination sites flanking two antiparallel ORFs in an arrangement which facilitates activation of one gene while repressing the other by Cre-dependent inversion of both ORFs.
The FLEX Cre-off switch consists of two pairs of heterotypic LoxP-variant recombination sites, namely LoxP having the wild type sequence and Lox2272 having a mutated sequence, flanking an ORF. Both LoxP variants are recognized by Cre, but only identical pairs of LoxP sites can recombine with each other and not with any other variant. The LoxP and Lox2272 sites are organized in an alternating fashion, with an antiparallel orientation for each pair. In the absence of Cre recombinase, the ORF can be expressed under the control of the user-selected promoter. In the presence of Cre, the LoxP and Lox2272 sites undergo recombination with the other LoxP and Lox2272 sites respectively, resulting in the inversion of the ORF to an antisense orientation and excision of one from each pair of identical recombination sites. Inversion of the ORF prevents expression of the gene of interest. Since the ORF is now flanked by two different LoxP-variant sites, no further recombination events will take place even when Cre is present.
An AAV 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. For the AAV (FLEX) conditional Cre-Switch gene expression vector, the FLEX Cre-Switch described above is placed in-between the two ITRs during vector construction, which is introduced into target cells along with the rest of viral genome. Expression of the second ORF in the FLEX Cre-Switch can then be activated while silencing the first ORF in the presence of Cre recombinase, upon Cre-mediated inversion of both ORF sequences.
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. Due to their low immunogenicity in host organisms, AAV is the ideal viral vector for many animal studies.
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.
|AAV1||Smooth muscle, CNS, lung, retina, pancreas, heart, liver|
|AAV2||Smooth muscle, CNS, liver, kidney, retina|
|AAV3||Smooth muscle, liver, lung|
|AAV4||CNS, retina, lung, kidney|
|AAV5||Smooth muscle, CNS, lung, retina|
|AAV6||Smooth muscle, heart, lung, adipose, liver|
|AAV7||Smooth muscle, retina, CNS, liver|
|AAV8||Smooth muscle, CNS, retina, liver, pancreas, heart, kidney, adipose|
|AAV9||Smooth muscle, lung, liver, heart, pancreas, CNS, retina, testes, kidney|
|AAVrh10||Smooth muscle, lung, liver, heart, pancreas, CNS, retina, kidney|
|AAV-DJ||Liver, heart, kidney, and spleen|
For further information about this vector system, please refer to the papers below.
|Methods in Enzy. 507:229-54 (2012)||Review of AAV virology and uses|
|Curr Opin Pharmacol. 24:59-67 (2015)||AAV use in gene therapy, and serotype differences|
|Gene. 216:55 (1998)||Characterization of LoxP mutants, including Lox2272|
|Nat Biotechnol. 21:562 (2003)||Development of the FLEX switch system|
|J Neurosci. 28:7025 (2008)||Application of a FLEX switch system|
The AAV (FLEX) conditional Cre-Switch gene expression vector is designed to achieve Cre-mediated switching between expression of two ORFs in mammalian cells and animals. Expression is under the control of a user-selected promoter and can be permanently switched from one user-selected ORF to another by co-expression of Cre recombinase.
This vector is optimized for high copy number replication in E. coli, high-titer packaging of live virus, efficient transduction of host cells, and high-level transgene expression. This AAV viral vector can be packaged into virus using all known capsid serotypes, is capable of very high transduction efficiency, and presents low safety risk.
Switch-like regulation: Opposite orientation of the two ORFs ensures that while the ORF in the sense orientation is expressed, the ORF in the antisense orientation is repressed without any leaky gene expression.
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: While our vector is mostly used for in vitro transduction of cultured cells, it can also be used to transduce cells in live animals. It is particularly suitable for the generation of transgenic animals with Cre-mediated conditional gene expression.
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, which leaves ~4.1 kb cargo space for the user's DNA of interest in the AAV (FLEX) conditional Cre-Switch gene expression vector.
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.
Promoter: The promoter driving your gene of interest is placed here.
Lox2272: Recombination site for Cre recombinase. Mutated Lox site with two base substitutions of wild type LoxP. Incompatible with LoxP sites. When Cre is present, the LoxP and LoxP2272 sites will be cut and recombine with compatible sites.
LoxP: Recombination site for Cre recombinase. Incompatible with Lox2272 sites. When Cre is present, the LoxP and Lox2272 sites will be cut and recombine with compatible sites.
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 #1: The open reading frame of a gene of interest is placed here, in a sense orientation. This gene can be expressed without Cre-mediated recombination.
ORF #2: The open reading frame of a gene of interest is placed here, in an antisense orientation. This gene can only be expressed after Cre-mediated recombination.
Regulatory element: Allows the user to add the Woodchuck hepatitis virus posttranscriptional regulatory element (WPRE). WPRE enhances transcriptional termination in the 3' LTR during viral RNA transcription, which leads to higher levels of functional viral RNA in packaging cells and hence greater viral titer. It also enhances transcriptional termination during the transcription of the user's gene of interest on the vector, leading to their higher expression levels.
BGH pA: Bovine growth hormone polyadenylation signal. It facilitates transcriptional termination of the upstream ORF.
3' ITR: 3' inverted terminal repeat. See description for 5’ ITR.
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.