293 cells for adenoviral packaging

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Constructing recombinant adenovirus with In-Fusion Cloning technology

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 into E. 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.

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Phase contrast microscopy image of the Adeno-X 293 Cell Line

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During adenovirus amplification and rescue, Adeno-X 293 cells typically remain intact but round up and may detach from the plate

During adenovirus amplification and rescue, Adeno-X 293 cells typically remain intact but round up and may detach from the plate. These changes are collectively referred to as the cytopathic effect (CPE). These images show CPE following transfection of Adeno-X ZsGreen1 DNA into the Adeno-X 293 Cell Line.