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Fig. 1. Schematic diagram of the plasmids used for generation of a recombinant adenovirus expressing antisense TGF-ß1 mRNA. A, the plasmid clone pCMV-asTGF-ß1 was constructed by fusing the PvuII fragment of pET3d-rTGF-ß1 in antisense orientation into the expressing vector pEGFP-C1, which was depleted for the EGFP. The transgene harboring the CMV promoter, the asTGF-ß1-fragment, and the SV40-polyadenylation signal was transferred to the adenoviral shuttle vector p{Delta}E1sp1A. B, the integration of vector sequences from p{Delta}E1sp1A-CMV-asTGF-ß1 into the adenoviral backbone vector pJM17 was performed by in vitro homologous recombination in the human embryo kidney cell line 293, which constitutively expresses the E1 transactivators required for propagation of recombinant adenoviruses. In the schematic diagram, the adenoviral DNA sequences are shown as thick black lines, the bacterial vector sequences as thinner black lines, the sequences encoding TGF-ß1 or EGFP as thick dark gray lines, and the markers for ampicillin (Amp), kanamycin (Kan), and tetracycline (Tet) conferring antibiotic resistance in Escherichia coli as thick gray lines, respectively.