Figure 3
Construction of a replication-defective recombinant adenovirus expressing enhanced green fluorescent protein under transcriptional control of the human cytomegalovirus immediate-early gene 1 promoter/enhancer. (A) In the mammalian reporter vector pEGFP-C1 the enhanced green fluorescent protein (EGFP) is expressed under control of the human cytomegalovirus immediate-early gene 1 promoter (PCMV). Abbreviations are: Kan/Neo, kanamycin/neomycin resistance genes; Amp, ampicillin resistance gene; pUC ori, E. coli origin of replication; SV40 ori, simian virus 40 origin of replication; SV40 polyA, simian virus 40 polyadenylation signal; HSVTK polyA, herpes simplex thymidine kinase polyadenylation signal. The adenoviral shuttle vector pΔE1sp1A contains Ad5 sequences from bp 22 (0 mu) to bp 5790 (16.1 mu) with a deletion of E1 sequences (ΔE1) from bp 342 to bp 3523 (1.0 - 9.8 mu) and a selectable ampicillin resistance gene (Amp). A multiple cloning site (MCS) containing unique restriction sites for Cla I, Bam HI, Xho I, Xba I, Eco RV, Eco RI, Hind III, Sal I, and Bgl II is embedded in the Ad5-sequences. For construction shuttle vector pΔE1sp1A-CMV-EGFP the 1656-bp Asn I/Ssp I fragment from plasmid pEGFP-C1 was filled in by Klenow DNA polymerase and cloned into the Eco RI digested and filled in pΔE1sp1A vector.(B) For integration of the reporter cassette from pE1Δsp1A-CMV-EGFP into the Ad5 backbone plasmid vector pJM17 both plasmids were cotransfected into human embryo kidney cell line 293 leading to homologous recombination between common Ad5 regions. Generation of recombinant viral particles were visualized by an increase of EGFP-positive cells and by viral focus formation in fluorescence microscopy. (C) Released replication-defective viral particles are infectious and are capable to deliver the CMV-EGFP cassette to target cells. The nucleotide sequence of the cloned vector pΔE1sp1A-CMV-EGFP is deposited in GenBank (Accession number AF288620).