FIG. 3.

Verification of the integrity of the bicistronic mRNA by RNase protection assay. (A) Schematic representation of monocistronic vector pVC (construct 1) and bicistronic vectors pCVC and pCVC1 (constructs 2 and 3) used to generate RNA templates. The regions A′, B′, and C′, protected by the three antisense RNA probes A, B, and C, are indicated. The RNA probes A, B, and C are slightly longer than the protected fragments because of the presence of additional nucleotides in the polylinker regions of the plasmids used as templates for the probes (see Materials and Methods). (B) Vectors shown in panel A were transfected in COS-7 cells. Total mRNAs were purified and analyzed by RNase A and T₁ protection (see Materials and Methods), using the RNA probes A (1054 nt), B (748 nt), and C (572 nt), complementary to nt 1 to 1046, 1 to 745, and 475 to 1046, respectively. The first lane corresponds to a mix of the three probes alone, without RNase treatment. The RNA templates and probes used are indicated at the bottom. The fragments protected by the probes A, B, and C are notated as A′ (1,017 nt), B′ (690 nt), and C′ (535 nt), respectively.