By SIRION Biotech…
SIRION recombinant adenoviruses for transient gene expression
Recombinant adenoviruses have become the viral vectors of choice where transient gene expression and quantitative gene regulation or knockdown is required. Adenoviruses from SIRION Biotech enable genetic engineering free of cell type limitations.
Adenovirus vectors are non-toxic, non-integrating, non-enveloped viruses with a linear double-stranded DNA. Adenovirus vectors providing strong, transient gene expression or -knockdown are particularly valuable in gene therapy, cancer therapy, and vaccine development.
Classical approaches to identification and validation of new targets in drug discovery use functional gene analysis in overexpression and knockdown studies. For many years, these studies were performed on cancer cell lines that are easy to handle and, more recently, on primary cells and even patient derived cells. Now classical transfection of cell lines has reached its limits.
Adenoviruses provide an ideal new research channel for finding new targets, primarily because adenovirus transduction is transient due to episomal genome expression, thus circumventing the risk of site-specific mutagenesis. Further, adenoviruses are characterized by high cell type accessibility without species limitations, and exhibit excellent quantitative transduction efficiency and high titer productions, enabling transgene delivery in vivo.
SIRION adenovirus production
SIRION’s adenovirus vector system with patented BAC technology uses a range of off-the-shelf engineered adenovirus vectors to enable recombinant adenovirus construction in less than five weeks.
The standard SIRION Biotech adenovirus service comprises:
- Cloning cDNA of Gene of Interest (GOI)/ shRNA up to 7.5 kb in an adenovirus shuttle plasmid to achieve adenovirus transfection
- DNA-Sequencing verification of cloning success
- Generation of recombinant adenovirus BACs
- Production of low passage virus stocks and amplification in HEK293 cells
- Purification and titration
- Gene expression quantification on mRNA level by qRT-PCR
This customizable adenovirus production system and generation service allows for virtually every desired gene or shRNA expression construct. Beside monocistronic and bicistronic expression systems, individual promoters or reporter genes can be chosen.
SIRION adenoviral vectors are based on the Ad5 serotype and use the Coxsackie-Adenovirus Receptor (CAR) to enter cells. To increase the packaging capacity in recombinant vectors the E1 and E3 genes are deleted from the wild type adenovirus. E1 is provided by the packaging cells (HEK293) and E3 is not essential for virus production. Deletion of E1 from the viral vector prevents viral replication to guarantee the safety of adenoviral vector.
The platform’s generous maximum packaging capacity of 7.5 kb creates space for multiple gene expression cassettes alongside series of other benefits:
- Strong, tightly regulated and highly efficient transient gene overexpression and knockdown in vitro and in vivo
- Reliable gene delivery into dividing and non-dividing cells with close to 100% efficiency
- Fast results – first gene expression within 24 hours
- High titer productions for transgene delivery in vivo
- Strong ubiquitous and inducible promoters and custom promoters
- Transduction enhancers for hard-to-transduce cell types
- No host genome integration
Algorithmic identification of shRNA sequences
shRNA is an artificial RNA molecule used to silence target gene expression via RNA interference (RNAi) by binding to the complementary target messenger mRNA in cells, to achieve gene silencing. Different viral vectors can be used to bring shRNA into cells, including adenoviruses that do not integrate into the genome, maintaining transient gene expression.
SIRION’s RNAiONE™ technology combines sophisticated algorithms with engineered viral vectors to define the most effective small hairpin RNA (shRNA) sequences for target gene identification and validation that can be further translated into SIRION’s viral vector platform.
Over the years SIRION Biotech has developed its base technology to the point where in 80% of recent projects it managed to identify targeted shRNA sequences with knockdown results of above 90%.
The applications of adenoviruses deliver the following benefits:
- Quantitative shRNA delivery and uniform gene expression in most cell types
- Highest possible gene expression levels and target gene knockdown
- Gene expression and level of knockdown adjustable over a wide range by virus amount
- Guaranteed knockdown of at least 80% on mRNA
- Cells tested to be virus-free allowing handling in S1 laboratories
- From cDNA sequence to functional gene expression cells in under six weeks
- Functionality tested frozen cells ready to use.
AdenoBOOST™ maximises adenovirus transfection
AdenoBOOST™ is a non-toxic peptide-based solution that specifically bridges the adenovirus surface to the cell membrane, enhancing gene expression by 20 to 50-fold in cell types which lack the primary adenovirus CAR-receptor. It can be used on almost any cell type without species limitations, and reduces the amount of virus particles needed per application.
Click on SIRION Biotech Adenovirus Services for more information.