Adenoviral Vector Generation Protocol
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Corresponding Organization : Nouscom (Italy)
Other organizations : University of Turin, University of Naples Federico II
Variable analysis
- Adenoviral vectors containing the coding sequences (CDS) for the transgenes
- Titer of each vector determined by qPCR and expressed as viral particles (vp) per mL
- HA tags added at the N- and C terminus of each transgene
- CDS for all the constructs generated by Gibson assembly and cloned into the respective shuttle plasmid containing the CMV promoter with two TetO operator repeats and a BGH polyA
- Expression cassettes transferred into pGAd plasmid, containing the E1/E3/E4 deleted in which the E4 is replaced with Ad5 E4 ORF6
- Transgene cassettes introduced in the E1 deletion locus of related pAdeno by homologous recombination in BJ5183 cells
- GAd vectors produced by transfection of adherent M9 cells (293 cells derivative) with Lipofectamine 2000 and amplification in suspension M9 cells
- Vectors purified from infected cells by Vivapure Adenopack 20 RT
- Positive control: Not explicitly mentioned
- Negative control: Not explicitly mentioned
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