By SIRION Biotech…
SIRION Biotech Transient Adenovirus Development
Recombinant adenoviruses have become the viral vectors of choice where transient transfection and quantitative gene regulation is required.
SIRION’s custom adenovirus production platform is based on a patented bacterial artificial chromosome (BAC) technology that enables construction in less than five weeks.
In addition, SIRION offers a range of off-the-shelf adenoviruses custom promoters as well as AdenoBOOST™ transduction enhancer for hard to transduce cell types such as NIH-3T3. Then used in combination with Ad-GFP, this can enhance adenovirus transduction up to 50-fold by bridging adenoviruses with the cell surface.
Overcoming transfection limits
Classical approaches for identification and validation of new targets in drug discovery use functional gene analysis through overexpression and knockdown approaches. For many years, this has mainly relied 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.
Adenovirus transduction provides ideal research channels to identify new targets, for three main reasons.
- Adenovirus transduction is transient due to episomal expression, thus circumventing the risk of site-specific mutagenesis.
- Adenoviruses are characterized by high accessibility without cell type limitations.
- Adenoviruses exhibit excellent quantitative transduction efficiency and high titer productions, enabling transgene delivery in vivo.
Transient adenovirus development
SIRION Biotech`s patented adenoviral vector platform allows for transient gene modification by offering a reliable system for highest gene overexpression or knockdown from vector cloning to functionally tested and purified viral particles or ready-to-use premade adenoviral particles.
A customized adenovirus vector system allows for virtually every desired expression construct. Beside monocistronic and bicistronic expression systems, individual promoters or reporter genes can be chosen.
SIRION Biotech adenovirus products have been successfully used in two main applications:
- Transient gene overexpression of the GOI in U87MG cells transduced with an Ad-CMV-TargetX vector, using Western Blot gene expression was detected three to five days after transduction, relative to cells transduced with an empty control vector (Ad-ctrl). This approach delivers benefits that include: quantitative gene delivery and uniform expression in most cell types, highest possible transient gene expression levels that are adjustable over a wide range of cells.
- Transient Gene Silencing using 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. Here, benefits also include quantitative shRNA delivery and uniform expression in most cell types, and guaranteed knockdown of at least 80% on mRNA.
Click on SIRION Biotech Adenovirus Design and Manufacture for more information.