Adenovirus FLEX Conditional Gene Expression Vector (Cre-Switch)

Overview

The adenovirus FLEX conditional Cre-Switch gene expression vector combines VectorBuilder’s highly efficient adenoviral vector system with the Cre-responsive FLEX conditional gene expression system to help you achieve adenovirus-mediated introduction of FLEX switch into a variety of mammalian cell types 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-Switch system consists of two pairs of heterotypic LoxP-variant recombination sites, namely LoxP, having the wild type sequence and Lox2272, having a mutated sequence flanking a pair of ORFs. 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 two ORFs are in an opposite orientation with respect to one-another, such that one ORF is in its proper sense orientation, while the other is in an antisense orientation. The LoxP and Lox2272 sites are organized in an alternating fashion, with an antiparallel orientation for each pair. In the absence of Cre recombinase, while the first ORF is expressed under the control of the user-selected promoter, the second ORF is not expressed due to its antisense orientation. 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 both ORFs and excision of one from each pair of identical recombination sites. Inversion of the ORFs results in silencing of the first ORF (which will now be in an antisense orientation) and allows expression of the second ORF (which will now be in a sense orientation).

An adenoviral vector is first constructed as a plasmid in E. coli. It is then transfected into packaging cells, where the region of the vector between the two inverted terminal repeats (ITRs) is packaged into live virus. For the adenovirus FLEX conditional Cre-Switch gene expression vector, the FLEX Cre-Switch described above is placed in between the two ITRs. When the virus is added to target cells, the DNA cargo is delivered into cells where it enters the nucleus and remains as episomal DNA without integration into the host genome. The FLEX Cre-Switch placed in-between the two ITRs during vector construction 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 turned on while silencing the first ORF in the presence of Cre recombinase, upon Cre-mediated inversion of both ORF sequences.

By design, adenoviral vectors lack the E1A, E1B and E3 genes (delta E1 + delta E3). The first two are required for the production of live virus (these two genes are engineered into the genome of packaging cells). As a result, virus produced from the vectors have the important safety feature of being replication incompetent (meaning that they can transduce target cells but cannot replicate in them).

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

References Topic
Proc Natl Acad Sci U S A. 91:8802 (1994) The 2nd generation adenovirus vectors
J Gen Virol. 36:59 (1977) A packaging cell line for adenovirus vectors
J Virol. 79:5437 (2005) Replication-competent adenovirus (RCA) formation in 293 Cells
Gene Ther. 3:75 (1996) A cell line for testing RCA
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

Highlights

The adenovirus 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 derived from the adenovirus serotype 5 (Ad5). It 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.

Advantages

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.

Low risk of host genome disruption: Upon transduction into host cells, adenoviral 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.

Very high viral titer: After our adenoviral vector is transfected into packaging cells to produce live virus, the virus can be further amplified to very high titer by re-infecting packaging cells. This is unlike lentivirus, MMLV retrovirus, or AAV, which cannot be amplified by re-infection. When adenovirus is obtained through our virus packaging service, titer can reach >1011 plaque-forming unit per ml (PFU/ml).

Broad tropism: Cells from commonly used mammalian species such as human, mouse and rat can be transduced with our vector, but some cell types have proven difficult to transduce (see disadvantages below).

Large cargo space: The upper limit size of the adenovirus genome for efficient virus packaging is ~38.7 kb (from 5' ITR to 3' ITR). After excluding the required backbone components for adenovirus gene expression and Cre-mediated recombination, our vector has about ~7.4 kb of cargo space to accommodate the user's DNA of interest. This is bigger than the ~6.3 kb cargo space in our lentiviral conditional gene expression vector and is sufficient for most applications.

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. It is particularly suitable for the generation of transgenic animals with Cre-mediated conditional gene expression.

Safety: The safety of our vector is ensured by the fact that it lacks genes essential for virus production (these genes are engineered into the genome of packaging cells). Virus made from our vector is therefore replication incompetent except when it is used to transduce packaging cells.

Disadvantages

Non-integration of vector DNA: The adenoviral genome does not integrate into the genome of transduced cells. Rather, it exists as episomal DNA, which can be lost over time, especially in dividing cells. 

Difficulty transducing certain cell types: While our adenoviral vectors can transduce many different cell types including non-dividing cells, it is inefficient against certain cell types such as endothelia, smooth muscle, differentiated airway epithelia, peripheral blood cells, neurons, and hematopoietic cells.

Strong immunogenicity: Live virus from adenoviral vectors can elicit strong immune response in animals, thus limiting certain in vivo applications.

Technical complexity: The use of adenoviral 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.

Key components

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.

Ψ: Adenovirus packaging signal required for the packaging of viral DNA into virus.

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.

TK pA: Herpes simplex virus thymidine kinase polyadenylation signal. It facilitates transcriptional termination of the upstream ORF.

ΔAd5: Portion of Ad5 genome between the two ITRs minus the E1A, E1B and E3 regions.

3' ITR: 3' inverted terminal repeat.

pBR322 ori: pBR322 origin of replication. Plasmids carrying this origin exist in medium copy numbers in E. coli.

Ampicillin: Ampicillin resistance gene. It allows the plasmid to be maintained by ampicillin selection in E. coli.

PacI: PacI restriction site (PacI is a rare cutter that cuts at TTAATTAA). The two PacI restriction sites on the vector can be used to linearize the vector and remove the vector backbone from the viral sequence, which is necessary for efficient packaging.

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