Takara Adeno-X GoStix \20 Tests

Adeno-X GoStix are designed to instantly confirm the presence of adenovirus in cell culture supernatants or lysates. The test detects adenoviral hexon proteins and can be used to determine if infectious virus was produced post-transfection, or for selecting the best time to harvest your virus. The included Hexon Control provides confirmation of GoStix test function.

Constructing recombinant adenovirus with In-Fusion Cloning technology.DNA sequences can be rapidly transferred as PCR products to any pAdenoX vector using the In-Fusion cloning method. In this example, your gene of interest is amplified with 15 bp extensions that are homologous to the ends of the linearized adenoviral vector. The PCR product is then purified and mixed with the linearized adenoviral vector of choice in the In-Fusion reaction. Following the reaction, a portion of the mixture is transformed intoE. coli(Stellar Competent Cells) and screened. Once a PCR-positive clone is identified, the recombinant pAdenoX vector is amplified, purified, and subsequently linearized with the restriction enzyme PacI, then transfected into Adeno-X 293 cells for viral rescue and amplification. Adeno-X GoStix can be used to determine the status of adenovirus rescue.

Early detection of adenovirus during rescue.Panel A. The Adeno-X GoStix protocol takes only a couple of minutes to indicate the presence of adenovirus particles. Supernatant from transfected cells is placed on the cassette, followed by chase buffer.Panel B. Adeno-X GoStix were used to confirm virus production during the rescue procedure. PacI-digested adenovirus expressing ZsGreen1 was transfected into HEK 293 cells, which were monitored for the emergence of cytopathic effects by phase and fluorescence microscopy (middle row). 20 μl of culture medium was applied to the Adeno-X GoStix on each day (bottom row). Four days post-transfection, Adeno-X GoStix were able to detect virus in culture supernatants containing plaques that were only weakly perceptible under phase microscopy and confirmed by fluorescence microscopy.

Rapid adenovirus detection during amplification.Panel A. Adenovirus expressing ZsGreen1 was amplified in HEK293 cells. 48 hr postinfection, and each day thereafter, the supernatant and the cell pellet were harvested, and titers were quantified by counting fluorescent cells in an infectious unit (IFU) assay. The increase in virus level in the cell pellets was mirrored by a proportional increase in the amount of virus collected from the culture supernatant. The amount of virus in the supernatant remained < 10% of the total virus output.Panel B. Supernatant serves as a clear indicator of total yields. In addition to IFU determination, 20 μl of each sample was applied to an Adeno-X GoStix. The increase in band intensity paralleled the increase in virus production observed by infectious unit titer.

What to expect when measuring viral titer with Lenti-X or Adeno-X GoStix.The control band (C) will always be detected, the test band shows (T) positive only you if there is enough virus in your sample.