AAV Conditional Gene Knockout Vector (Floxed)

The AAV conditional gene knockout vector combines VectorBuilder’s highly versatile AAV vector system with the Cre-mediated conditional gene knockout system to help you achieve AAV-mediated in vitro and in vivo delivery of Cre-mediated inactivation of gene expression. This floxed system comprises LoxP recombination sites flanking a gene of interest to facilitate inactivation of gene expression by Cre-dependent deletion of the coding sequence. In the absence of Cre recombinase, expression of the gene of interest is permitted. When Cre is introduced into cells carrying this vector, the gene of interest is permanently excised.

An AAV vector is first constructed as a plasmid in E. coli where a LoxP-flanked gene of interest is cloned in-between two inverted terminal repeats (ITRs). 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. When AAV virus is added to target cells, gene expression can be inactivated in the presence of Cre recombinase upon Cre-mediated excision of the coding sequence.

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. 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.

Note this vector alone is not sufficient for achieving recombination between pairs of LoxP sites. Coexpression of Cre is required either via a helper vector or mRNA encoding Cre. For further information about this vector system and Cre-mediated recombination, please refer to the papers below.

The AAV conditional gene knockout vector is designed to achieve Cre-mediated conditional gene deletion in mammalian cells and animals. Expression of the gene of interest is initially under the control of the user-selected promoter and can be permanently silenced by co-expression of Cre recombinase, which will permanently excise the region encoding for the gene of interest. 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.

Stable gene inactivation: Treatment with Cre recombinase will permanently remove the sequence encoding the gene of interest and prevents its transcription.

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

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 conditional gene knockout 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.

LoxP: Recombination site for Cre recombinase. When Cre is present the region flanked by the two LoxP sites will be excised.

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 posttranscriptional 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.

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.