SIRION Biotech Adenovirus Services

Recombinant adenoviruses have become the viral vectors of choice when transient and quantitative gene regulation is required. Adenoviruses from SIRION Biotech enable genetic engineering free of cell type limitations.

SIRION’s custom adenovirus construction service is based on a patented bacterial artificial chromosome (BAC) technology that enables construction in less than five weeks.

In addition, SIRION offers custom promoters and transduction enhancers for hard to transduce cell types as well as a range of off-the-shelf adenoviruses.

Background

Classical approaches to identifying and validating new targets in drug discovery use functional gene analysis using overexpression and knockdown studies. For many years, the field had to mainly rely on cancer cell lines that are easy to handle and manipulate.

More recently, the need for maximum authenticity has shifted the focus to primary cells and even patient derived cells. Now, however, classical transfection of those cells is also reaching its limits.

Adenoviruses provide an ideal new research channel for identifying target genes, for three main reasons.

• Adenovirus transduction is transient due to episomal genome expression, thus circumventing the risk of site-specific mutagenesis
• Adenoviruses are characterized by high cell type accessibility without species limitations
• Adenoviruses exhibit excellent quantitative transduction efficiency and high titer productions

Product features

SIRION Biotech`s patented adenoviral vector platform allows for transient gene modification by offering a reliable system for highest gene overexpression or knockdown. It enables you to go from vector cloning to a functional tested and purified virus stocks in less than four weeks or ready-to-use frozen and functionally tested cell pools in less than six weeks.

A customized adenovirus vector generation service allows for virtually every desired expression construct. Beside the standard multicistronic expression systems, individual promoters or reporter genes can be chosen.

Adenovirus services

The standard SIRION Biotech adenovirus service comprises:

• Cloning the cDNA of the Gene of Interest (GOI) up to 7.5 kb/ shRNA in an adenovirus shuttle plasmid
• DNA-Sequencing verification of cloning success
• Generation of recombinant adenovirus BACs
• Production of low passage virus stocks and amplification in 293 cells
• Purification and titration
• Gene expression quantification on mRNA level by qRT-PCR

Optional services

In addition, customers can specify:

Transduction of customer cells and freezing 48h after transduction.
Gene expression quantification on protein level by Western Blot and quality control.
Shipment of ready-to-use viruses/ transduced cells along with detailed product documentation.

Additional services

SIRION Biotech also offers off-the-shelf premade and validated adenoviruses, an adenovirus transduction enhancer and products summarized under the AdenoONE™ brand.

All included, SIRION Biotech offers the most comprehensive portfolio for adenovirus applications.

Adenovirus Applications

SIRION Biotech adenovirus products have been successfully used in two main applications:

Transient Gene Overexpression
Transient expression of the GOI in U87MG cells transduced with an Ad-CMV-TargetX vector, typically using Western Blot to show gene expression three to five days after transduction, relative to cells transduced with an empty control vector (Ad-ctrl).

This application of adenoviruses delivers the following benefits:

• Quantitative gene delivery and uniform expression in most cell types
• Highest possible gene expression levels
• Gene expression adjustable over a wide range by virus amount (copy number)
• 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

Transient Gene Silencing
This uses transient knockdown of CDK5 in HUVEC cells transduced with the Ad-U6-shRNA vector. Again, Western Blot or qRT-PCR is used to show CDK5 expression four to eight days after transduction relative to NT-shRNA expressing control cells.

Use of adenoviruses in this application delivers benefits that include:

• Quantitative shRNA delivery to and uniform expression in most cell types
• Highest possible target gene knockdown
• Degree of Knockdown adjustable over a wide range by virus amount
• Guaranteed knockdown of at least 80% on mRNA when combined with RNAiONETM
• Cells tested to be virus-free allowing handling in S1 laboratories
• Delivery of functionality tested knockdown cells in less than six weeks
• Functionality tested frozen cells ready to use

In Vivo Applications
Adenoviruses can be used in vivo in cases that include:

• Stereotactic injection to specific brain regions
• Intravenous liver injections
• Ex-vivo transduction and re-implantation in regenerative medicine

Hard to transduce cell types

Challenging cell types such as NIH-3T3 can be transduced with Ad-GFP either with or without the SIRION Biotech transduction enhancer AdenoBOOST. This can enhance adenovirus transduction up to 50-fold by bridging adenoviruses with the cell surface.