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Transduction

Transduction of mammalian cells using an adenoviral vector
(here rAV-LifeAct)

The term “transduction” is used to describe a virus-mediated DNA transfer into cells. In contrast to transfection of cells with foreign DNA or RNA, no transfection reagent is needed here. The viral vector, itself, also called virion, is able to infect cells and transport the DNA directly into the nucleus, independent of further actions. After the release of the DNA into the nucleus, the protein of interest is produced using the cells’ own machineries. 

Adenoviral vectors have proven to be a very successful tool in many different human and rodent cell types. Transduction efficiencies of up to 100% can easily be achieved. Moreover, this method also gives access to difficult-to-transfect cells, such as primary cells.

With the rAV-LifeAct adenoviral vectors, ibidi provides a perfect tool for visualizing F-actin in difficult-to-access cells (e.g., endothelial cells) without interfering with the cytoskeletal dynamics. 

Method Comparison

Transfection

Adenoviral Transduction

Transient and stable protein expression

Only transient protein expression

Easy-to-transfect cells (e.g., cell lines)

Difficult-to-transfect cells (e.g., primary cells)

Variable transfection efficiency

Up to 100% transduction efficiency

Biosafety level 1

Biosafety level 2

Transduction of Primary Endothelial Cells

Human umbilical vein endothelial cells (HUVEC) were transduced with the adenoviral vector rAV CMV-LifeAct-TagRFP and cultured under flow conditions (20 dyn / cm²) in a  µ-Slide I 0.4 Luer over 9 days.

Adenoviruses have a large genome of 36kb, making genetic manipulations by classical cloning strategies extremely difficult and ineffective. The ibiClone Adeno Adenoviral Cloning Kit enables fast and easy recombination-based adenoviral vector generation in E.coli cells, followed by adenovirus production in HEK 293 cells in under 4 weeks.

The purity of adenovirus preparations is an important key to successful adenovirus transduction, since cellular debris and medium-derived proteins can cause toxic effects. Moreover, they can also affect viral activity during long-term storage. The ibiPure Adeno Adenovirus Purification Kit enables fast and easy chromatography-based adenovirus purification within only 20 minutes, directly from the cell pellet.

The restricted uptake of viruses often lowers the success of gene or shRNA expression, respectively, and requires the application of higher viral titers. However, increasing the amount of viruses to boost expression often leads to toxic side effects in many cell types. ibidi’s ibiBoost Adeno Adenovirus Transduction Enhancer was developed to specifically enhance the efficiency of the virus transfer into difficult to transduce mammalian and rodent cells. By bridging the virus surface to the cell membrane, the virus uptake is significantly increased without toxicity.