• FAQs: Transfection with rAdenovirus

    In this section you will find information and commonly asked Q&As pertaining to Adenovirus use.

    Click on the following links for more information:
        ► Transfection efficiency
        ► High expression of recombinant proteins with recombinant adenovirus
        ► Comparison between adenovirus, lentivirus & AAV
        ► Q&A: Premade Recombinant Adenovirus of Human Full-length cDNA

    High transfection efficiency

    Adenovirus can be used to transfect a wide range of cells including primary cells and stem cells as well animal models, such as monkeys, mouse and human cells. Adenoviral transfection efficiency can reach 100%, compared to 30% with lentivirus and conventional plasmids. For hard to be transfected cells, recombinant adenovirus could dramatically increase the transfection efficiency.

    Premade Adenovirus Level Comparison

    Figure: Transfection efficiency is much higher of recombinant adenovirus when compared to plasmid transfection.

    High expression of recombinant proteins with recombinant adenovirus

    Proteins expressed in adenovirus have identical folding and post-translational modifications as native proteins, with expression level being very high, often reaching 10-20% of total cellular proteins.

    Premade Adenovirus Western Data

    Figure: Protein Expression in HEK293t cells when recombinant adenovirus transduction compared to plasmid transfecton.
    Protein Expression is much higher in HEK293t cells when they were transfected with 3µg plasmid DNA and recombinant adenovirus carried the same gene at multiplicity of infections (MOI, 50:1). 72hrs post-transfection and 48hrs post-adenovirus transduction, HEK293 cells were harvested and lysed. 10µg total protein were separated with SDS-PAGE and overexpressed proteins were then detected with anti-flag M2 antibody(Sigma).

    A comparison between adenovirus, lentivirus & AAV

    Comparison of three major gene therapy viral vectors with large insert

    A Comparsion Between Adeno, Lenti and AAV

    Comparing three major gene therapy viral vectors, recombinant adenovirus can carry much large insert. It can achieve high transduction efficiency and protein expression level. After initial packaging, it is easy to reproduce, purify and concentrate to high titer. In comparison to lentivirus, recombinant adenovirus is very stable.

    Q&A: Premade Recombinant Adenovirus of Human Full-length cDNA

    Is recombinant adenovirus safe?
    Adenoviral vector is replication deficient and consider to be safe and can be handled in BSL2 labs. Premade Recombinant Adenovirus are with E1 and E3 gene deletion, they are unable to replicate except in the packaging cell line. They should be treated like other recombinant DNA materials, for example, plasmid clones.

    Is gene expression from recombinant adenovirus transient or stable?
    When gene is delivered into mammalian cells by recombinant adenovirus, the expression is transient. Unlike lentivirus, recombinant adenoviruses genome remain epichromosomal in host cells, results transient gene expression.

    Is recombinant adenoviruses toxic to host cells?
    Recombinant adenovirus may trigger immunoresponse in vivo, but it shows no toxicity to transducted cells in vitro. Post-transfection viability of the host cells is almost 100%; it is well tolerated in a wide range of cells. Unlike plasmid transfection, toxic chemicals have to be used during transfection.

    What is the largest transgene can accommodated by recombinant adenovirus in ViGene Biosciences' human full-length cDNA collection?
    Our E1 & E3 deletion adenoviral vector can hold up to 8kb gene insertion. Although large transgene often shows decreased protein expression, we can detect 6.5kb transgene expression in transducted HEK293 cells.

    Is recombinant adenovirus stable?
    Recombinant adenoviruses can be stored at fridge for weeks, -20℃ freezers for months and -80℃ freezers for years.

    How easy to propagate recombinant adenovirus from ViGene Biosciences?
    Once an initial stock is generated, the adenovirus can be easily amplified in HEK293 cells to achieve very high titer. Each mammalian cell can produce 10,000 adenoviruses. For example, a 10cm culture dish can produce 1X1011 viruses.

    What promoter drives human full-length cDNA expression in ViGene Biosciences' recombinant adenovirus?
    Strong CMV promoter in our vector enables the transgene protein to accumulate at very high levels. Our adenoviral system is an ideal mammalian expression system for hard- to- express proteins including membrane proteins.

    What does ViGene Biosciences guarantee in human full-length cDNA expression with recombinant adenovirus?
    We guarantee the human full-length cDNA expression in HEK293 cells by western blot. We validated the expression of thousands of our recombinant adenovirus. If any our premade recombinant adenovirus failed to express the right protein by our customer, we will validated in HEK203 cells. If we failed, we will refound our customer.

    Is the premade recombinant adenovirus second day delivery?
    All of our premade recombinant adenovirus are second day delivery. However, due to long term storage in -80℃, the viral titer might be different with our label and the volume of delivery is 100ul. To satisfy our customers, we can propagate the recombinant adenovirus to 500ul at the titer higher than 1010vp/ml without of charge in 5 days.