Figure 2.

RNA aptamer expression system based on double infection with recombinant vaccinia viruses. (A) Design of the T7-RNA expression cassette. (B) Schematic representation of the vaccinia virus-based cytoplasmatic RNA aptamer expression system. (C) Course of the expression of TR-encoded aptamer in vT7-coinfected Jurkat E6 cells shown by a representative dot-blot analysis for aptamer TR-D31 (Right). Maximum levels of aptamer expression are seen 7 h postinfection. Quantification (Left) was done as follows: total cellular RNA was isolated, transferred to the blotting membrane and hybridized with 5′-³²P-labeled oligonucleotide complementary to the 3′-stabilizing stem-loop structure present in all TR constructs. For comparison and quantification, in vitro-transcribed aptamer TR-D20 was treated in the same way. D, Dot blot analysis and quantification of maximally expressed aptamer RNA. Each dot was quantified on a PhosphorImager. Quantification was done as described for C.