Figure 3

Serial deletions in U3 and in the transcriptional control region and functional analysis of transcriptional readthrough. A. The U3 deletion mutagenesis strategy and oligonucleotide primer list. The PCR strategy used for the generation of nlCre reporter constructs is illustrated. The first PCR product, generated by EF1α and a mutagenesis primer, was used as a mega-primer in a second PCR with the IRES primer to generate the DNA fragment for nlCre plasmid cloning. All mutagenesis constructs were verified by DNA sequencing. The amplified products were cloned into pdl-EF-3'LTR-IRES-nlCre (Fig. 1). B. and C. The 3' LTR deletion series. Genetic structure of the LTR U3 is illustrated at the top diagram. The deletion clones were generated in pBluescript subclones verified by squencing before being swapped into the final reporter construct. The reporter constructs were verified again by sequencing. TE26 cells were transfected with 0.1 or 0.2 μg of different plasmid DNA and 48 h later, fixed and stained with 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside, and the RNA readthrough rate was determined. The bar graphs to the right summarize the results of RNA readthrough analysis. The results shown are representative of more than three independent duplicate or triplicate transfections.