| Viral genome modification | To insert the Kozak sequence before E1A, a KpnI fragment from pShuttle-DM-E2F-Δ-24, containing the E2F promoter and E1A, was subcloned into pGEM-3Z (Promega). This plasmid was then used to replace the E1A translation start site with oligonucleotides containing the Kozak sequence. The KpnI fragment, containing the E2F-E1A modified with the Kozak sequence, was returned to pShuttle-DM-E2F-Δ-24 to generate pShuttle-DM-E2F-KD24. Finally, pShuttle-DM-E2F-Δ-24 was recombined by homologous recombination with pVK503, which contains the complete Ad5 genome with an RGD-modified fiber. |